JP2024515169A - Cancer treatment using NK cells and CD20-targeted antibodies - Google Patents

Abstract

The present invention provides, inter alia, methods of treating patients suffering from CD20+ cancers.

Description

PRIORITY CLAIM This application claims priority to U.S. Provisional Application No. 63/290,349, filed December 16, 2021, and U.S. Provisional Application No. 63/172,401, filed April 8, 2021, the entire contents of which are incorporated herein by reference.

Targeted therapies, including antibody therapy, have revolutionized cancer treatment. One mechanism by which antibody therapy induces cytotoxicity is through antibody-dependent cell-mediated cytotoxicity (ADCC). Many cancer patients may not mount a strong ADCC response. A reduced ADCC response may significantly reduce the effectiveness of any given monoclonal antibody therapeutic agent in the patient, leading to non-response or relapse. Thus, a reduced ADCC response may negatively impact clinical outcomes.

Despite the recent discovery and development of several anti-cancer drugs, the poor prognosis of many types of cancer, including CD20+ cancers, continues to require improved methods and therapeutic agents.

The present invention solves these and other problems in the art.

NK cells are immune cells that can engage tumor cells not only through their complex array of cell surface receptors, but also through antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells can engage antibodies through their surface CD16 receptor to initiate ADCC. NK cells have advantages over other immune cells, such as T cells, used in CAR-T cell therapy and other cell therapies. One exemplary advantage is that NK cells can be used as an allogeneic therapy, where NK cells from a donor can be safely used in one or more patients without HLA matching, gene editing or other genetic manipulation. Allogeneic NK cells with anti-tumor activity can be safely administered to patients without many of the risks associated with T cell therapy, such as severe cytokine release syndrome (CRS) and neurotoxicity or graft-versus-host disease (GvHD).

Allogeneic NK cells can provide a significant treatment option for cancer patients. One exemplary advantage is that NK cells have excellent tolerability without the onset of graft-versus-host disease, neurotoxicity, or cytokine release syndrome associated with other cell-based therapies. Another exemplary advantage is that NK cells do not require prior antigen exposure or expression of specific antigens to identify and lyse tumor cells. Another exemplary advantage is that NK cells have the unique ability to link innate immunity and mount a multi-clonal adaptive immune response to build long-term anti-cancer immune memory. All of these characteristics contribute to the efficacy of NK cells as a potential cancer treatment option.

For example, NK cells can recruit and activate other components of the immune system. Activated NK cells secrete cytokines and chemokines, such as interferon gamma (IFNγ); tumor necrosis factor alpha (TNFα); and macrophage inflammatory protein 1 (MIP1), which signal to recruit T cells to tumors. NK cells also directly kill tumor cells, thereby exposing tumor antigens for recognition by the adaptive immune system.

In conjunction with this, by using a diverse cord blood bank as a source of NK cells, cord blood with favorable characteristics (e.g., high affinity CD16 and killer cell immunoglobulin-like receptor (KIR) B-haplotype) can be selected to enhance clinical activity.

As described herein, administration of allogeneic NK cells can enhance a patient's ADCC response, for example, during the course of monoclonal antibody therapy.

Thus, the present invention provides, among other things, a method for treating patients suffering from CD20+ cancer.

The present invention provides a method of treating a patient suffering from a CD20+ cancer comprising administering a population of natural killer cells (NK cells) and an antibody that targets human CD20, where the NK cells are allogeneic to the patient, have a KIR-B haplotype, and are homozygous for a CD16 158V polymorphism.

In some embodiments, the cancer is non-Hodgkin's lymphoma (NHL). In some embodiments, the NHL is an indolent NHL. In some embodiments, the indolent NHL is selected from the group consisting of follicular lymphoma, lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia, gastric MALT, non-gastric MALT, lymph node marginal zone lymphoma, splenic marginal zone lymphoma, small cell lymphocytic lymphoma (SLL) and chronic lymphocytic lymphoma (CLL). In some embodiments, the small cell lymphocytic lymphoma (SLL) or chronic lymphocytic lymphoma (CLL) includes lymph node or splenic involvement. In some embodiments, the NHL is an aggressive NHL. In some embodiments, the aggressive NHL is selected from the group consisting of diffuse large B-cell lymphoma, mantle cell lymphoma, transformed follicular lymphoma, follicular lymphoma (stage IIIB), transformed mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal large B-cell lymphoma, lymphoblastic lymphoma, and aggressive B-cell lymphoma with MYC and BCL2 translocations. In some embodiments, the aggressive B-cell lymphoma with MYC and BLC2 translocations additionally contains a BCL6 translocation.

In some embodiments, the patient has relapsed after treatment with an anti-CD20 antibody. In some embodiments, the patient has experienced disease progression after treatment with autologous stem cell transplant or chimeric antigen receptor T-cell therapy (CAR-T).

In some embodiments, the patient is administered 1×10 ⁸ to 1×10 ¹⁰ NK cells. In some embodiments, the patient is administered 1×10 ⁹ to 8×10 ⁹ NK cells. In some embodiments, the patient is administered 4×10 ⁸ , 1×10 ⁹ , 4×10 ⁹ or 8×10 ⁹ NK cells.

In some embodiments, the antibody is selected from the group consisting of obinutuzumab (or a biosimilar thereof), ofatumumab (or a biosimilar thereof), or a combination thereof. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody is ofatumumab.

In some embodiments, the patient undergoes lymphodepleting chemotherapy prior to treatment. In some embodiments, the lymphodepleting chemotherapy is non-myeloablative chemotherapy. In some embodiments, the lymphodepleting chemotherapy comprises treatment with one or more of cyclophosphamide and fludarabine. In some embodiments, the lymphodepleting chemotherapy comprises treatment with cyclophosphamide and fludarabine. In some embodiments, the cyclophosphamide is administered at 100-500 mg/m ² /day. In some embodiments, the cyclophosphamide is administered at 250 mg/m ² /day. In some embodiments, the cyclophosphamide is administered at 500 mg/m ² /day. In some embodiments, the fludarabine is administered at 10-50 mg/m ² /day. In some embodiments, fludarabine is administered at 30 mg/m ² /day.

In some embodiments, the method further comprises administration of IL-2. In some embodiments, the IL-2 is administered to the patient at 1×10 ⁶ IU/m ^2. In some embodiments, the IL-2 is administered to the patient at 6,000,000 IU. In some embodiments, the administration of IL-2 occurs within 1-4 hours of administering the NK cells.

In some embodiments, the administration of NK cells and human CD20 targeted antibody occurs weekly. In some embodiments, the administration of NK cells and human CD20 targeted antibody occurs weekly for 4-8 weeks. In some embodiments, the administration of NK cells occurs weekly and the administration of human CD20 targeted antibody occurs every other week.

In some embodiments, the NK cells are not genetically modified. In some embodiments, at least 70% of the NK cells are CD56+ and CD16+. In some embodiments, at least 85% of the NK cells are CD56+ and CD3-. In some embodiments, 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+, and 1% or less of the NK cells are CD14+. In some embodiments, each administration of NK cells is administration of between 1×10 ⁹ and 5×10 ⁹ NK cells. In some embodiments, each administration of NK cells is administration of between 1×10 ⁹ and 5×10 ⁹ NK cells. In some embodiments, prior to the first administration of NK cells, a dose of a CD20-targeted antibody is administered to the patient.

In some embodiments, the expanded natural killer cells are expanded cord blood natural killer cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% CD16+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKG2D+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp46+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp30+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% DNAM-1+ cells. In some embodiments, the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp44+ cells. In some embodiments, the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less or 0% CD3+ cells. In some embodiments, the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less or 0% CD14+ cells. In some embodiments, the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD19+ cells. In some embodiments, the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD38+ cells.

In some embodiments, the natural killer cells do not contain a CD16 transgene. In some embodiments, the natural killer cells do not express exogenous CD16 protein. In some embodiments, the expanded natural killer cells are not genetically engineered.

In some embodiments, the expanded natural killer cells are derived from the same cord blood donor.

In some embodiments, the population of NK cells comprises at least 100 million expanded natural killer cells, e.g., 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 90 billion, 100 billion, 200 billion, 250 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion.

In some embodiments, the population of NK cells is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; and (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, a TNFα mutant and a 4-1BBL gene to produce expanded natural killer cells, thereby producing a population of expanded natural killer cells.

In some embodiments, the population of NK cells is produced by a method comprising: (a) obtaining seed cells comprising natural killer cells from umbilical cord blood; (b) depleting the seed cells of CD3+ cells; (c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, TNFα mutation and the 4-1BBL gene to produce a master cell bank population of expanded natural killer cells; and (d) expanding the master cell bank population of expanded natural killer cells by culturing a second plurality of Hut78 cells engineered to express membrane-bound IL-21, TNFα mutation and the 4-1BBL gene to produce expanded natural killer cells; thereby producing a population of expanded natural killer cells.

In some embodiments, the population of NK cells is produced by a method that, after step (c), further comprises: (i) freezing the master cell bank population of amplified natural killer cells in a plurality of containers; and (ii) thawing the containers containing aliquots of the master cell bank population of amplified natural killer cells, wherein in step (d), amplifying the master cell bank population of amplified natural killer cells comprises amplifying the aliquots of the master cell bank population of amplified natural killer cells.

In some embodiments, the cord blood is from a donor who is KIR-B haplotype and homozygous for the CD16 158V polymorphism.

In some embodiments, the population of NK cells is produced by a method comprising expanding natural killer cells from umbilical cord blood at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold or 70,000-fold.

In some embodiments, the expanded population of natural killer cells is not enriched or sorted after expansion. In some embodiments, the percentage of NK cells expressing CD16 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKG2D in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp30 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp44 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood. In some embodiments, the percentage of NK cells expressing NKp46 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood. In some embodiments, the percentage of NK cells expressing DNAM-1 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in the seed cells derived from umbilical cord blood.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this application are incorporated herein by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims.

Incorporation by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

The novel features of the invention are set forth with particularity in the appended claims. The patent or application file contains one or more drawings executed in color. Copies of this patent or patent application publication containing color drawings will be provided by the Patent and Trademark Office upon request and payment of the appropriate fee. The features and advantages of the present invention will be better understood with reference to the following detailed description which sets forth illustrative embodiments in which the principles of the invention are applied, and the accompanying drawings, in which:

FIG. 1 shows an exemplary embodiment for a method for expanding and stimulating NK cells. FIG. 1 shows that umbilical cord blood-derived NK cells (CB-NK) have approximately 10-fold higher culture expansion potential than peripheral blood-derived NK cells (PB-NK) in preclinical experiments. FIG. 1 shows that expression of tumor-engaging NK-activating immunoreceptors is higher and more consistent in cord blood-derived products compared to peripheral blood sources. FIG. 1 shows the phenotype of expanded and stimulated NK cell populations. FIG. 1 shows the key steps of manufacturing AB-101 drug product as an example of a cord blood-derived NK cell expanded population. FIG. 1 shows the purity of AB-101 (n=9). FIG. 1 shows the purity of CD3-depleted cells, MCB and DP produced under GMP conditions. FIG. 1 shows the expression of NK cell receptors in CD3-depleted cells, MCB and DP, produced under GMP conditions. FIG. 1 shows NK purity (CD56+/CD3−) as determined by flow cytometry. FIG. 1 shows CD38+ expression in expanded NK cells from three cord blood donors. FIG. 1 shows the mean CD38+ fluorescence intensity of CD38+ NK cells from three cord blood donors. FIG. 1 shows differential surface protein expression in NK cell sources compared to AB-101 cells as starting material. FIG. 1 shows that obinutuzumab significantly induces ADCC of AB-101 cells versus ARH-77.

The present invention provides, inter alia, natural killer (NK) cells, e.g., expanded and stimulated NK cells, methods for producing NK cells, pharmaceutical compositions comprising NK cells, and methods for treating patients, e.g., suffering from cancer, with NK cells.

I. Expansion and Stimulation of Natural Killer Cells In some embodiments, natural killer cells are expanded and stimulated, for example, by culture and stimulation with feeder cells.

NK cells can be expanded and stimulated, for example, as described in US 2020/0108096 or WO 2020/101361, both of which are incorporated herein by reference in their entireties. Briefly, source cells can be cultured on recombinant HuT-78 (ATCC® TIB-161 ^™ ) cells engineered to express 4-1BBL, membrane-bound IL-21, and ^mutant TNFα, as described in US 2020/0108096.

Suitable NK cells can also be expanded and stimulated as described herein.

In some embodiments, the NK cells are expanded and stimulated by a method comprising: (a) providing NK cells, e.g., a composition comprising NK cells, e.g., CD3(+) depleted cells; and (b) culturing in a medium comprising feeder cells and/or stimulatory factors to produce a population of expanded and stimulated NK cells.

A. Natural Killer Cell Sources In some embodiments, the NK cell source is selected from the group consisting of peripheral blood, peripheral blood lymphocytes (PBLs), peripheral blood mononuclear cells (PBMCs), bone marrow, umbilical cord blood (umbilical cord blood or cord blood), isolated NK cells, NK cells derived from induced pluripotent stem cells, NK cells derived from embryonic stem cells, and combinations thereof.

In some embodiments, the NK cell source is a single unit of umbilical cord blood.

In some embodiments, a natural killer cell source, for example, a single cord blood unit, comprises 1×10 ⁷ or about 1×10 ⁷ to 1×10 ⁹ or about 1×10 ⁹ total nucleated cells. In some embodiments, a natural killer cell source, for example, a single cord blood unit, comprises 1×10 ⁸ or about 1×10 ⁸ to 1.5×10 ⁸ or about 1.5×10 ⁸ total nucleated cells. In some embodiments, a natural killer cell source, for example, a single cord blood unit, comprises 1×10 ⁸ total nucleated cells. In some embodiments, a natural killer cell source, for example, a single cord blood unit, comprises about 1×10 ⁸ total nucleated cells. In some embodiments, a natural killer cell source, for example, a single cord blood unit, comprises 1×10 ⁹ total nucleated cells. In some embodiments, a natural killer cell source, for example, a single unit of umbilical cord blood, contains about 1 x ¹⁰⁹ total nucleated cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises about 20% to about 80% CD16+ cells. In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises about 20% to about 80%, about 20% to 70%, about 20% to 60%, about 20% to 50%, about 20% to 40%, about 20% to 30%, about 30% to 80%, about 30% to 70%, about 30% to 60%, about 30% to % to 50% or about 50%, about 30% to 40% or about 40%, about 40% to 80% or about 80%, about 40% to 70% or about 70%, about 40% to 60% or about 60%, about 40% to 50% or about 50%, about 50% to 80% or about 80%, about 50% to 70% or about 70%, about 50% to 60% or about 60%, about 60% to 80% or about 80%, about 60% to 70% or about 70%, or about 70% to 80% or about 80%. In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 80% or less CD16+ cells. Alternatively, some NK cell sources may comprise a concentration of CD16+ cells greater than 80%.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, MLG2A+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, contains 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, NKG2C+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, NKG2D+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, contains 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, NKp46+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, contains 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, NKp30+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, contains 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, DNAM-1+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, contains 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, NKp44+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, of CD25+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, of CD62L+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, CD69+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, contains 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, CXCR3+ cells.

In some embodiments, the NK cell source, e.g., umbilical cord blood unit, comprises 40% or less, e.g., 30% or less, e.g., 20% or less, e.g., 10% or less, e.g., 5% or less, of CD57+ cells.

In some embodiments, the NK cells in the NK cell source comprise a KIR B allele of the KIR receptor system. See, e.g., Hsu et al., "The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism," Immunological Review 190:40-52 (2002); and Pyo et al. , "Different Patterns of Evolution in the Centromeric and Telemeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-like Receptor Locus," PLoS One5: e15115 (2010).

In some embodiments, the NK cells in the NK source contain the 158V/V variant of CD16 (i.e., homozygous CD16 15V polymorphism). See, e.g., Koene et al., "FcγRIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L/R/H Phenotype," Blood 90:1109-14 (1997).

In some embodiments, the NK cells in the cell source contain both the KIR B allele of the KIR receptor system and the 158V/V variant of CD16.

In some embodiments, the NK cells in the cell source are not genetically engineered.

In some embodiments, the NK cells in the cell source do not contain a CD16 transgene.

In some embodiments, the NK cells in the cell source do not express exogenous CD16 protein.

In some embodiments, the NK cell source is CD3(+) depleted. In some embodiments, the method includes depleting the NK cell source of CD3(+) cells. In some embodiments, depleting the NK cell source of CD3(+) cells includes contacting the NK cell source with a CD3-binding antibody or antigen-binding fragment thereof. In some embodiments, the CD3-binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1 and HIT3a, and fragments thereof. In some embodiments, the CD3-binding antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is attached to beads, e.g., magnetic beads. In some embodiments, depleting the CD3(+) cells in the composition includes contacting the composition with a CD3-targeting antibody or antigen-binding fragment thereof attached to beads to remove bead-bound CD3(+) cells from the composition. The composition can be CD3 depleted by immunomagnetic selection, for example, using the CliniMACS T cell depletion set (LS Depletion set (162-01) Miltenyi Biotec).

In some embodiments, the NK cell source is enriched for CD56+, for example, by gating on CD56 expression.

In some embodiments, the NK cell source is CD56+ enriched and CD3(+) depleted, for example, by selecting CD56+CD3- expressing cells.

In some embodiments, the NK cell source contains both the KIR B allele of the KIR receptor system and the 158V/V variant of CD16, e.g., CD56+ enrichment and CD3(+) depletion are achieved by sorting for CD56+CD3- expressing cells.

B. Feeder Cells The present invention discloses feeder cells for expanding NK cells. These feeder cells advantageously allow the expansion of NK cells to numbers suitable for producing the pharmaceutical compositions described herein. In some cases, the feeder cells allow the expansion of NK cells, often with cell expansion on other types of feeder cells or using other methods, without a decrease in CD16 expression. In some cases, the feeder cells allow the expanded NK cells to better tolerate freezing, such that a higher proportion of NK cells remain viable after a freeze/thaw cycle, or the cells remain viable for a longer period during freezing. In some cases, the feeder cells allow the NK cells to maintain high levels of cytotoxicity such as ADCC, prolong survival, increase persistence, and enhance or maintain high levels of CD16. In some cases, the feeder cells allow the NK cells to expand without inducing significant levels of exhaustion or senescence.

Feeder cells can be used to stimulate NK cells and help them expand more quickly, for example by providing substrates, growth factors and/or cytokines.

NK cells can be stimulated using various types of feeder cells, including, but not limited to, peripheral blood mononuclear cells (PBMC), Epstein-Barr virus-transformed B-lymphoblast cells (e.g., EBV-LCL), myeloid leukemia cells (e.g., K562) and CD4(+) T cells (e.g., HuT), and derivatives thereof.

In some embodiments, the feeder cells are inactivated, for example, by gamma-irradiation or mitomycin-c treatment.

Feeder cells suitable for use in the methods described herein are described, for example, in US 2020/0108096, the entire contents of which are incorporated herein by reference.

In some embodiments, the feeder cells are inactivated CD4(+) T cells. In some embodiments, the inactivated CD4(+) T cells are HuT-78 cells (ATCC® TIB-161 ^™ ), or variants or derivatives thereof. In some embodiments, the HuT-78 derivative is H9 ( ^{ATCC® HTB} -176 ^™ ).

In some embodiments, the inactivated CD4(+) T cells express OX40L. In some embodiments, the inactivated CD4(+) T cells are HuT-78 cells that express OX40L (SEQ ID NO: 4), or a variant thereof, or a variant or derivative thereof.

In some embodiments, the feeder cells are HuT-78 cells engineered to express one or more genes selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2) and mutant TNFα (SEQ ID NO:3) ("eHut-78 cells"), or variants thereof.

In some embodiments, the inactivated CD4(+) T cells are HuT-78 ( ^ATCC® TIB-161 ^™ ) cells or variants or derivatives expressing an OX40L ortholog, or variants thereof. In some embodiments, the feeder cells are HuT-78 cells engineered to express one or more genes selected from the group consisting of a 4-1BBL ortholog, or variants thereof, a membrane-bound IL-21 ortholog, or variants thereof, and a mutant TNFα ortholog, or variants thereof.

In some embodiments, the feeder cells are HuT-78 cells that express OX40L (SEQ ID NO: 4); and have been engineered to express 4-1BBL (SEQ ID NO: 1), membrane-bound IL-21 (SEQ ID NO: 2) and mutant TNFα (SEQ ID NO: 3) ("eHut-78 cells"), or variants or derivatives thereof.

In some embodiments, the feeder cells are expanded, e.g., from frozen stocks, prior to culturing with the NK cells, e.g., as described in Example 2.

C. Stimulating factor

NK cells can also be stimulated with one or more other stimulatory factors other than feeder cells, e.g., signaling factors, in addition to or instead of feeder cells.

In some embodiments, the stimulatory factor, e.g., signal transduction factor, is a component of the culture medium, as described herein. In some embodiments, the stimulatory factor, e.g., signal transduction factor, is a supplement to the culture medium, as described herein.

In some embodiments, the stimulatory factor is a cytokine. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, IL-23, IL-27, IFN-α, IFN-β, and combinations thereof.

In some embodiments, the cytokine is IL-2.

In some embodiments, the cytokine is a combination of IL-2 and IL-15.

In some embodiments, the cytokines are a combination of IL-2, IL-15 and IL-18.

In some embodiments, the cytokines are a combination of IL-2, IL-18 and IL-21.

D. Cultivation

NK cells can be expanded and stimulated by co-culturing the NK cell source with feeder cells and/or other stimulatory factors. Suitable NK cell sources, feeder cells and stimulatory factors are described herein.

In some cases, the resulting expanded population of natural killer cells is enriched and/or sorted after expansion. In some cases, the resulting expanded population of natural killer cells is not enriched and/or sorted after expansion.

The present invention also describes compositions that include the various culture compositions described herein, e.g., NK cells. For example, a composition is described that includes an expanded population of cord blood-derived natural killer cells that include a KIR-B haplotype and a CD16 158V polymorphism homozygote, and a plurality of engineered HuT78 cells.

The invention also describes a vessel, e.g., a vial, cryobag, etc., in which the resulting expanded population of natural killer cells is contained. In some cases, a plurality of vessels contain a portion of the resulting expanded population of natural killer cells, including at least 10 vessels, e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 vessels.

The present invention also describes a bioreactor containing various culture compositions, e.g., NK cells, as described herein. For example, the bioreactor contains a culture comprising natural killer cells derived from a natural killer cell source as described herein, and feeder cells as described herein. The present invention also describes a bioreactor containing the resulting expanded population of natural killer cells.

1. Culture Media The present invention discloses culture media for expanding NK cells. These culture media advantageously allow the expansion of NK cells to a cell number suitable for producing the pharmaceutical compositions described herein. In some cases, the culture media allows the expansion of NK cells without loss of CD16 expression, often accompanied by cell expansion on other helper cells or in other media.

In some embodiments, the culture medium is a basal culture medium, optionally supplemented with additional components, e.g., as described herein.

In some embodiments, the culture medium, e.g., basal culture medium, is a serum-free culture medium. In some embodiments, the culture medium, e.g., basal culture medium, is a serum-free culture medium supplemented with human plasma and/or serum.

Suitable basal culture media include DMEM, RPMI 1640, MEM, DMEM/F12, SCGM (CellGenix®, ^20802-0500 or 20806-0500), LGM-3 ^™ (Lonza, CC-3211), TexMACS ^™ (Miltenyi Biotec, 130-097-196), ALyS ^™ 505NK-AC (Cell Science and Technology Institute, Inc., 01600P02), ALyS ^™ 505NK-EX (Cell Science and Technology Institute, Inc., 01400P10), CTS Examples of suitable optical analyzers include, but are not limited to, ^the AIM-V ^™ SFM (ThermoFisher Scientific, A3830801), the CTS ^™ OpTmizer ^™ (ThermoFisher Scientific, A1048501, ABS-001, StemXxVivo), and combinations thereof.

The culture medium may contain additional components or may be supplemented with additional components such as growth factors, signaling factors, nutrients, antigen binding agents, etc. The culture medium may be supplemented by introducing the respective additional component or components into the culture vessel prior to, simultaneously with, or after the medium is added to the culture vessel. The additional components or components may be added together or separately. If added separately, the additional components do not need to be added simultaneously.

In some embodiments, the culture medium includes plasma, e.g., human plasma. In some embodiments, the culture medium is supplemented with plasma, e.g., human plasma. In some embodiments, the plasma, e.g., human plasma, includes an anti-coagulant, e.g., trisodium citrate.

In some embodiments, the medium comprises and/or is supplemented with plasma, e.g., human plasma, at 0.5% or about 0.5% to 10% or about 10% v/v. In some embodiments, the medium is 0.5% or about 0.5% to 9% or about 9%, 0.5% or about 0.5% to 8% or about 8%, 0.5% or about 0.5% to 7% or about 7%, 0.5% or about 0.5% to 6% or about 6%, 0.5% or about 0.5% to 5% or about 5%, 0.5% or about 0.5% to 4% or about 4%, 0.5% or about 0.5% to 3% or about 3%, 0.5% or about 0.5% to 2% or about 2%, 0.5% or about 0.5% to 1% or about 1%, 1% or about 1% to 10% or about 10%, 1% or about 1% to 9% or about 9%, 1% or about 1% to 8% or about 8%, 1% or about 1% to 7% or about 7%, 1% or about 1% to 6% or about 6%, 1% or about 1% to 5% or about 5%, 1% or about 1% to 4% or about 4%, 1% or about 1% to 3% or about 3%, 1% or about 1% to 2% or about 2%, 2% or about 2% to 10% or about 10%, 2% or about 2% to 9% or about 9%, 2% or about 2% to 8% or about 8%, 2% or about 2% to 7% or about 7%, 2% or about 2% to 6% or about 6%, 2% or about 2% to 5% or about 5%, 2% or about 2% to 4% or about 4%, 2% or about 2% to 3% or about 3%, 3% or about 3% to 10% or about 10%, 3% or about 3% to 9% or about 9%, 3% or about 3% to 8% or about 8%, 3% or about 3% to 7% or about 7%, 3% or about 3% to 6% or about 6%, 3% or about 3% to 5% or about 5%, 3% or about 3% to 4% or about 4%, 4% or about 4% to 10% or about 10%, 4% or about 4% to 9% or about 9%, 4% or about 4% to 8% or about 8%, 4% or about 4% to 7% or about 7%, 4% or about 4% to 6% or about 6%, 4% or about 4% to 5% or about 5%, 5% or about 5% to 10% or about 10%, 5% or about 5% to 9% or about 9% , 4% or about 4% to 8% or about 8%, 5% or about 5% to 7% or about 7%, 5% or about 5% to 6% or about 6%, 6% or about 6% to 10% or about 10%, 6% or about 6% to 9% or about 9%, 6% or about 6% to 8% or about 8%, 6% or about 6% to 7% or about 7%, 7% or about 7% to 10% or about 10%, 7% or about 7% to 9% or about 9%, 7% or about 7% to 8% or about 8%, 8% or about 8% to 10% or about 10%, 8% or about 8% to 9% or about 9%, or 9% or about 9% to 10% or about 10% v/v of plasma, e.g., human plasma. In some embodiments, the culture medium comprises and/or is supplemented with 0.8% to 1.2% v/v of human plasma. In some embodiments, the culture medium comprises and/or is supplemented with 1.0% v/v human plasma. In some embodiments, the culture medium comprises and/or is supplemented with about 1.0% v/v human plasma.

In some embodiments, the culture medium includes serum, e.g., human serum. In some embodiments, the culture medium is supplemented with serum, e.g., human serum. In some embodiments, the serum is inactivated, e.g., heat inactivated. In some embodiments, the serum is filtered, e.g., sterile-filtered.

In some embodiments, the culture medium comprises glutamine. In some embodiments, the culture medium is supplemented with glutamine. In some embodiments, the culture medium comprises and/or is supplemented with 2.0 or about 2.0 to 6.0 or about 6.0 mM glutamine. In some embodiments, the culture medium comprises and/or is supplemented with 2.0 or about 2.0 to 5.5 or about 5.5, 2.0 or about 2.0 to 5.0 or about 5.0, 2.0 or about 2.0 to 4.5 or about 4.5, 2.0 or about 2.0 to 4.0 or about 4.0, 2.0 or about 2.0 to 3.5 or about 3.5, 2.0 or about 2.0 to 3.0 or about 3.0, 2.0 or about 2.0 to 2.5 or about 2.5, 2.5 or about 2.5 to 6.0 or about 6.0, 2.5 or about 2.5 to 6.0 or about 6.0, or about 2.5-5.5 or about 5.5, 2.5 or about 2.5-5.0 or about 5.0, 2.5 or about 2.5-4.5 or about 4.5, 2.5 or about 2.5-4.0 or about 4.0, 2.5 or about 2.5-3.5 or about 3.5, 2.5 or about 2.5-3.0 or about 3.0, 3.0 or about 3.0-6.0 or about 6.0, 3.0 or about 3.0-5.5 or about 5.5, 3.0 or about 3.0-5.0 or about 5.0, 3.0 or about 3.0- 4.5 or about 4.5, 3.0 or about 3.0-4.0 or about 4.0, 3.0 or about 3.0-3.5 or about 3.5, 3.5 or about 3.5-6.0 or about 6.0, 3.5 or about 3.5-5.5 or about 5.5, 3.5 or about 3.5-5.0 or about 5.0, 3.5 or about 3.5-4.5 or about 4.5, 3.5 or about 3.5-4.0 or about 4.0, 4.0 or about 4.0-6.0 or about 6.0, 4.0 or about 4.0-5.5 or 5.5, 4.0 or about 4.0-5.0 or about 5.0, 4.0 or about 4.0-4.5 or about 4.5, 4.5 or about 4.5-6.0 or about 6.0, 4.5 or about 4.5-5.5 or about 5.5, 4.5 or about 4.5-5.0 or about 5.0, 5.0 or about 5.0-6.0 or about 6.0, 5.0 or about 5.0-5.5 or about 5.5, or 5.5 or about 5.5-6.0 or about 6.0 mM. In some embodiments, the culture medium comprises and/or is supplemented with 3.2 mM to 4.8 mM glutamine. In some embodiments, the culture medium comprises and/or is supplemented with 4.0 mM glutamine. In some embodiments, the culture medium comprises and/or is supplemented with about 4.0 mM glutamine.

In some embodiments, the culture medium includes one or more cytokines. In some embodiments, the culture medium is supplemented with one or more cytokines.

In some embodiments, the cytokine is selected from IL-2, IL-12, IL-15, IL-18, and combinations thereof.

In some embodiments, the culture medium comprises and/or is supplemented with IL-2. In some embodiments, the culture medium comprises and/or is supplemented with IL-2 at 150 or about 150 to 2,500 or about 2,500 IU/ml. In some embodiments, the culture medium comprises and/or is supplemented with IL-2 at 200 or about 200 to 2,250 or about 2,250, 200 or about 200 to 2,000 or about 2,000, 200 or about 200 to 1,750 or about 1,750, 200 or about 200 to 1,500 or about 1,500, 200 or about 200 to 1,250 or about 1,250, 200 or about 200 to 1,000 or about 1,000, 200 or about 200-750 or about 750, 200 or about 200-500 or about 500, 200 or about 200-250 or about 250, 250 or about 250-2,500 or about 2,500, 250 or about 250-2,250 or about 2,250, 250 or about 250-2,000 or about 2,000, 250 or about 250-1,750 or about 1,750, 250 or about 250-1,500 or about 1,5 00, 250 or about 250-1,250 or about 1,250, 250 or about 250-1,000 or about 1,000, 250 or about 250-750 or about 750, 250 or about 250-500 or about 500, 500 or about 500-2,500 or about 2,500, 500 or about 500-2,250 or about 2,250, 500 or about 500-2,000 or about 2,000, 500 or about 50 0 to 1,750 or about 1,750, 500 or about 500 to 1,500 or about 1,500, 500 or about 500 to 1,250 or about 1,250, 500 or about 500 to 1,000 or about 1,000, 500 or about 500 to 750 or about 750, 750 or about 750 to 2,250 or about 2,250, 750 or about 750 to 2,000 or about 2,000, 750 or about 750 to 1,750 or about 1,750, 750 or about 750-1,500 or about 1,500, 750 or about 750-1,250 or about 1,250, 750 or about 750-1,000 or about 1,000, 1,000 or about 1,000-2,500 or about 2,500, 1,000 or about 1,000-2,250 or about 2,250, 1,000 or about 1,000-2,000 or about 2,000, 1,000 or about 1,000-1,750 or about 1,750, 1,000 or about 1,000-1,500 or about 1,500, 1,000 or about 1,000-1,250 or about 1,250, 1,250 or about 1,250-2,500 or about 2,500, 1,250 or about 1,250-2,250 or about 2,250, 1,250 or about 1,250-2,000 or about 2,000, 1,250 or about 1,250- 1,750 or about 1,750, 1,250 or about 1,250-1,500 or about 1,500, 1,500 or about 1,500-2,500 or about 2,500, 1,500 or about 1,500-2,250 or about 2,250, 1,500 or about 1,500-2,000 or about 2,000, 1,500 or about 1,500-1,750 or about 1,750, 1,750 or about 1,750-2,500 or about 2,500, 1,750 or about 1,750-2,250 or about 2,250, 1,750 or about 1,750-2,000 or about 2,000, 2,000 or about 2,000-2,500 or about 2,500, 2,000 or about 2,000-2,250 or about 2,250, or 2,250 or about 2,250-2,500 or about 2,500 IU/ml.

In some embodiments, the culture medium contains and/or is supplemented with 64 μg/l to 96 μg/l of IL-2. In some embodiments, the culture medium contains and/or is supplemented with 80 μg/l (about 1,333 IU/ml) of IL-2. In some embodiments, the culture medium contains and/or is supplemented with about 80 μg/l of IL-2.

In some embodiments, the culture medium contains and/or is supplemented with a combination of IL-2 and IL-15.

In some embodiments, the culture medium contains and/or is supplemented with a combination of IL-2, IL-15 and IL-18.

In some embodiments, the culture medium contains and/or is supplemented with a combination of IL-2, IL-18 and IL-21.

In some embodiments, the culture medium comprises and/or is supplemented with glucose. In some embodiments, the culture medium comprises and/or is supplemented with 0.5 or about 0.5-3.5 g/l or about 3.5 g/l of glucose. In some embodiments, the culture medium comprises and/or is supplemented with 0.5 or about 0.5-3.0 or about 3.0, 0.5 or about 0.5-2.5 or about 2.5, 0.5 or about 0.5-2.0 or about 2.0, 0.5 or about 0.5-1.5 or about 1.5, 0.5 or about 0.5-1.0 or about 1.0, 1.0 or about 1.0-3.0 or about 3.0, 1.0 or about 1.0-2.5 or about 2.5, 1.0 or about 1.0- 2.0 or about 2.0, 1.0 or about 1.0-1.5 or about 1.5, 1.5 or about 1.5-3.0 or about 3.0, 1.5 or about 1.5-2.5 or about 2.5, 1.5 or about 1.5-2.0 or about 2.0, 2.0 or about 2.0-3.0 or about 3.0, 2.0 or about 2.0-2.5 or about 2.5, or 2.5 or about 2.5-3.0 or about 3.0 g/l. In some embodiments, the culture medium comprises and/or is supplemented with 1.6-2.4 g/l of glucose. In some embodiments, the culture medium comprises and/or is supplemented with 2.0 g/l of glucose. In some embodiments, the culture medium comprises about 2.0 g/l of glucose.

In some embodiments, the culture medium includes and/or is supplemented with sodium pyruvate. In some embodiments, the culture medium includes and/or is supplemented with 0.1 or about 0.1 to 2.0 or about 2.0 mM sodium pyruvate. In some embodiments, the culture medium contains at least one selected from the group consisting of 0.1 or about 0.1-1.8 or about 1.8, 0.1 or about 0.1-1.6 or about 1.6, 0.1 or about 0.1-1.4 or about 1.4, 0.1 or about 0.1-1.2 or about 1.2, 0.1 or about 0.1-1.0 or about 1.0, 0.1 or about 0.1-0.8 or about 0.8, 0.1 or about 0.1-0.6 or about 0.6, 0.1 or about 0.1-0.4 or about 0.4, 0.1 or about 0.1-0.2 or about 0.2, 0.2 or about 0.2-2.0 or about 2.0, 0.2 or about 0.2-1.8 or about 1.8, 0.2 or about 0.2-1.6 or about 1.6, 0.2 or about 0.2-1.4 or about 1.4, 0.1 or about 0.1-1.2 or about 1.2, 0.2 or about 0.2-1.2 or about 1.2, 0.2 or about 0.2-1.0 or about 1.0, 0.2 or about 0.2-0.8 or about 0.8, 0.2 or about 0.2-0.6 or about 0.6, 0.2 or about 0.2-0.4 or about 0.4, 0.4 or about 0.4-2.0 or about 2.0, 0.4 or about 0.4-1.8 or about 1.8, 0.4 or is about 0.4 to 1.6 or about 1.6, 0.4 or about 0.4 to 1.4 or about 1.4, 0.4 or about 0.4 to 1.2 or about 1.2, 0.4 or about 0.4 to 1.0 or about 1.0, 0.4 or about 0.4 to 0.8 or about 0.8, 0.4 or about 0.4 to 0.6 or about 0.6, 0.6 or about 0.6 to 2.0 or about 2.0, 0.6 or about 0. 0.6 or about 0.6 to 1.6 or about 1.6, 0.6 or about 0.6 to 1.4 or about 1.4, 0.6 or about 0.6 to 1.2 or about 1.2, 0.6 or about 0.6 to 1.0 or about 1.0, 0.6 or about 0.6 to 0.8 or about 0.8, 0.8 or about 0.8 to 2.0 or about 2.0, 0.8 or about 0.8 to 1 0.8 or about 0.8-1.6 or about 1.6, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.2 or about 1.2, 0.8 or about 0.8-1.0 or about 1.0, 1.0 or about 1.0-2.0 or about 2.0, 1.0 or about 1.0-1.8 or is about 1.8, 1.0 or about 1.0-1.6 or about 1.6, 1.0 or about 1.0-1.4 or about 1.4, 1.0 or about 1.0-1.2 or about 1.2, 1.2 or about 1.2-2.0 or about 2.0, 1.2 or about 1.2-1.8 or about 1.8, 1.2 or about 1.2-1.6 or about 1.6, 1.2 or about 1.2-1.4 or about 1. 4, 1.4 or about 1.4-2.0 or about 2.0, 1.4 or about 1.4-1.8 or about 1.8, 1.4 or about 1.4-1.6 or about 1.6, 1.6 or about 1.6-2.0 or about 2.0, 1.6 or about 1.6-1.8 or about 1.8, or 1.8 or about 1.8-2.0 or about 2.0 mM. In some embodiments, the culture medium comprises 0.8-1.2 mM sodium pyruvate. In some embodiments, the culture medium comprises 1.0 mM sodium pyruvate. In some embodiments, the culture medium comprises about 1.0 mM sodium pyruvate.

In some embodiments, the culture medium comprises and/or is supplemented with sodium bicarbonate. In some embodiments, the culture medium comprises and/or is supplemented with 0.5 or about 0.5 to 3.5 or about 3.5 g/l of sodium bicarbonate. In some embodiments, the culture medium comprises and/or is supplemented with 0.5 or about 0.5 to 3.0 or about 3.0, 0.5 or about 0.5 to 2.5 or about 2.5, 0.5 or about 0.5 to 2.0 or about 2.0, 0.5 or about 0.5 to 1.5 or about 1.5, 0.5 or about 0.5 to 1.0 or about 1.0, 1.0 or about 1.0 to 3.0 or about 3.0, 1.0 or about 1.0 to 2.5 or about 2.5, 1.0 or about 1. 0-2.0 or about 2.0, 1.0 or about 1.0-1.5 or about 1.5, 1.5 or about 1.5-3.0 or about 3.0, 1.5 or about 1.5-2.5 or about 2.5, 1.5 or about 1.5-2.0 or about 2.0, 2.0 or about 2.0-3.0 or about 3.0, 2.0 or about 2.0-2.5 or about 2.5, or 2.5 or about 2.5-3.0 or about 3.0 g/l. In some embodiments, the culture medium comprises and/or is supplemented with 1.6-2.4 g/l of sodium bicarbonate. In some embodiments, the culture medium comprises and/or is supplemented with 2.0 g/l of sodium bicarbonate. In some embodiments, the culture medium comprises about 2.0 g/l of sodium bicarbonate.

In some embodiments, the culture medium comprises and/or is supplemented with albumin, e.g., human albumin, e.g., a human albumin solution described herein. In some embodiments, the culture medium comprises and/or is supplemented with a 20% albumin solution, e.g., 0.5% or about 0.5% to 3.5% or about 3.5% v/v of a 20% human albumin solution. In some embodiments, the culture medium comprises a 20% albumin solution, e.g., a 20% human albumin solution, at 0.5 or about 0.5% to 3.0 or about 3.0%, 0.5% or about 0.5% to 2.5 or about 2.5%, 0.5% or about 0.5% to 2.0 or about 2.0%, 0.5% or about 0.5% to 1.5 or about 1.5%, 0.5% or about 0.5% to 1.0 or about 1.0%, 1.0 or about 1.0% to 3.0 or about 3.0%, 1.0 or about 1.0% to 2.5 or about 2.5%, 1 0 or about 1.0% to 2.0 or about 2.0%, 1.0 or about 1.0% to 1.5 or about 1.5%, 1.5 or about 1.5% to 3.0 or about 3.0%, 1.5 or about 1.5% to 2.5 or about 2.5%, 1.5 or about 1.5% to 2.0 or about 2.0%, 2.0% or about 2.0% to 3.0 or about 3.0%, 2.0% or about 2.0% to 2.5 or about 2.5%, or 2.5% or about 2.5% to 3.0 or about 3.0% v/v. In some embodiments, the culture medium comprises and/or is supplemented with a 20% albumin solution, e.g., a 20% human albumin solution at 1.6% to 2.4% v/v. In some embodiments, the culture medium comprises and/or is supplemented with a 20% albumin solution, e.g., a 20% human albumin solution, at 2.0% v/v. In some embodiments, the culture medium comprises a 20% albumin solution, e.g., a 20% human albumin solution, at about 2.0% v/v.

In some embodiments, the culture medium comprises and/or is supplemented with 2 or about 2 to 6 or about 6 g/l of albumin, e.g., human albumin. In some embodiments, the culture medium comprises and/or is supplemented with 2 or about 2 to 5.5 or about 5.5, 2 or about 2 to 5.0 or about 5.0, 2 or about 2 to 4.5 or about 4.5, 2 or about 2 to 4 or about 4, 2 or about 2 to 3.5 or about 3.5, 2 or about 2 to 3 or about 3, 2 or about 2 to 2.5 or about 2.5, 2.5 or about 2.5 to 6 or about 6, 2.5 or about 2.5 to 5.5 or about 5.5, 2.5 or about 2.5 to 5.5 or about 5.5, 2.5 or about 2.5 to 5.0 or about 5.0, 2.5 or about 2.5 to 4.5 or about 4.5, 2.5 or about 2.5 to 4.0 or about 4.0, 2.5 or about 2.5 to 3.5 or about 3.5, 2.5 or about 2.5 to 3.0 or about 3.0, 3 or about 3 to 6 or about 6, 3 or about 3 to 5.5 or about 5.5, 3 or about 3-5 or about 5, 3 or about 3-4.5 or about 4.5, 3 or about 3-4 or about 4, 3 or about 3-3.5 or about 3.5, 3.5 or about 3.5-6 or about 6, 3.5 or about 3.5-5.5 or about 5.5, 3.5 or about 3.5-5 or about 5, 3.5 or about 3.5-4.5 or about 4.5, 3.5 or about 3.5-4 or about 4, 4 or about 4-6 or about 6, 4 or or about 4-5.5 or about 5.5, 4 or about 4-5 or about 5, 4 or about 4-4.5 or about 4.5, 4.5 or about 4.5-6 or about 6, 4.5 or about 4.5-5.5 or about 5.5, 4.5 or about 4.5-5 or about 5, 5 or about 5-6 or about 6, 5 or about 5-5.5 or about 5.5, or 5.5 or about 5.5-6 or about 6 g/l. In some embodiments, the culture medium comprises and/or is supplemented with 3.2-4.8 g/l of albumin, e.g., human albumin. In some embodiments, the culture medium comprises 4 g/l of albumin, e.g., human albumin. In some embodiments, the culture medium comprises about 4 g/l of albumin, e.g., human albumin.

In some embodiments, the culture medium is supplemented with Poloxamer 188. In some embodiments, the culture medium comprises and/or is supplemented with 0.1 or about 0.1-2.0 or about 2.0 g/l of Poloxamer 188. In some embodiments, the culture medium comprises and/or is supplemented with 0.1 or about 0.1-1.8 or about 1.8, 0.1 or about 0.1-1.6 or about 1.6, 0.1 or about 0.1-1.4 or about 1.4, 0.1 or about 0.1-1.2 or about 1.2, 0.1 or about 0.1-1.0 or about 1.0, 0.1 or about 0.1-0.8 or about 0.8, 0.1 or about 0.1-0.6 or about 0.6, 0.1 or about 0.1-0.4 or about 0.4, 0.1 or about 0.1-0.2 or about 0.2, 0.2 or about 0.2-2.0 or about 2.0, 0.2 or about 0.2-1.8 or about 1.8, 0.2 or about 0.2-1.6 or about 1.6, 0.2 or about 0.2-1.4 or about 1.4 , 0.2 or about 0.2-1.2 or about 1.2, 0.2 or about 0.2-1.0 or about 1.0, 0.2 or about 0.2-0.8 or about 0.8, 0.2 or about 0.2-0.6 or about 0.6, 0.2 or about 0.2-0.4 or about 0.4, 0.4 or about 0.4-2.0 or about 2.0, 0.4 or about 0.4-1.8 or about 1.8, 0.4 or about 0.4 to 1.6 or about 1.6, 0.4 or about 0.4 to 1.4 or about 1.4, 0.4 or about 0.4 to 1.2 or about 1.2, 0.4 or about 0.4 to 1.0 or about 1.0, 0.4 or about 0.4 to 0.8 or about 0.8, 0.4 or about 0.4 to 0.6 or about 0.6, 0.6 or about 0.6 to 2.0 or about 2.0, 0.6 or about 0 0.6 or about 0.6 to 1.8 or about 1.8, 0.6 or about 0.6 to 1.6 or about 1.6, 0.6 or about 0.6 to 1.4 or about 1.4, 0.6 or about 0.6 to 1.2 or about 1.2, 0.6 or about 0.6 to 1.0 or about 1.0, 0.6 or about 0.6 to 0.8 or about 0.8, 0.8 or about 0.8 to 2.0 or about 2.0, 0.8 or about 0.8 to 1 0.8 or about 0.8-1.6 or about 1.6, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.4 or about 1.4, 0.8 or about 0.8-1.2 or about 1.2, 0.8 or about 0.8-1.0 or about 1.0, 1.0 or about 1.0-2.0 or about 2.0, 1.0 or about 1.0-1.8 or is about 1.8, 1.0 or about 1.0-1.6 or about 1.6, 1.0 or about 1.0-1.4 or about 1.4, 1.0 or about 1.0-1.2 or about 1.2, 1.2 or about 1.2-2.0 or about 2.0, 1.2 or about 1.2-1.8 or about 1.8, 1.2 or about 1.2-1.6 or about 1.6, 1.2 or about 1.2-1.4 or about 1. 4, 1.4 or about 1.4-2.0 or about 2.0, 1.4 or about 1.4-1.8 or about 1.8, 1.4 or about 1.4-1.6 or about 1.6, 1.6 or about 1.6-2.0 or about 2.0, 1.6 or about 1.6-1.8 or about 1.8, or 1.8 or about 1.8-2.0 or about 2.0 g/l. In some embodiments, the culture medium comprises 0.8-1.2 g/l of Poloxamer 188. In some embodiments, the culture medium comprises 1.0 g/l of Poloxamer 188. In some embodiments, the culture medium comprises about 1.0 g/l of Poloxamer 188.

In some embodiments, the culture medium includes and/or is supplemented with one or more antibiotics.

An exemplary first culture medium is shown in Table 1.

Exemplary second culture media are listed in Table 2.

2. CD3 Binding Antibody In some embodiments, the culture medium comprises and/or is supplemented with a CD3 binding antibody or antigen-binding fragment thereof. In some embodiments, the CD3 binding antibody or antigen-binding fragment thereof is selected from the group consisting of OKT3, UCHT1 and HIT3a, or a mutant thereof. In some embodiments, the CD3 binding antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof.

In some embodiments, the CD3 binding antibody or antigen-binding fragment thereof and the feeder cells are added to the culture vessel prior to the addition of the NK cells and/or culture medium.

In some embodiments, the culture medium comprises and/or is supplemented with OKT3 at 5 or about 5 ng/ml to 15 or about 15 ng/ml. In some embodiments, the culture medium comprises and/or is supplemented with OKT3 at 5 or about 5 to 12.5 or about 12.5, 5 or about 5 to 10 or about 10, 5 or about 5 to 7.5 or about 7.5, 7.5 or about 7.5 to 15 or about 15, 7.5 or about 7.5 to 12.5 or about 12.5, 7.5 or about 7.5 to 10 or about 10, 10 or about 10 to 15 or about 15, 10 or about 10 to 12.5 or about 12.5, or 12.5 or about 12.5 to 15 or about 15 ng/ml. In some embodiments, the culture medium comprises and/or is supplemented with OKT3 at 10 ng/ml. In some embodiments, the culture medium contains and/or is supplemented with OKT3 at about 10 ng/ml.

3. Culture Vessels A variety of vessels are contemplated within the teachings of the present invention. In some embodiments, the culture vessel is selected from the group consisting of a flask, a bottle, a dish, a multi-well plate, a roller bottle, a bag, and a bioreactor.

In some embodiments, the culture vessel is treated to be hydrophilic. In some embodiments, the culture vessel is treated to promote attachment and/or proliferation. In some embodiments, the surface of the culture vessel is coated with serum, collagen, laminin, gelatin, poly-L-lysine, fibronectin, extracellular matrix proteins, and combinations thereof.

In some embodiments, different types of culture vessels are used for different culture stages.

In some embodiments, the culture vessel has a volume of 100 ml or about 100 ml to 1,000 l or about 1,000 l. In some embodiments, the culture vessel has a volume of 125 ml or about 125 ml, 250 ml or about 250 ml, 500 ml or about 500 ml, 1 l or about 1 l, 5 l or about 5 l, 10 l or about 10 l, or 20 l or about 20 l.

In some embodiments, the culture vessel is a bioreactor.

In some embodiments, the bioreactor is a rocking bed (wave motion) bioreactor. In some embodiments, the bioreactor is a stirred tank bioreactor. In some embodiments, the bioreactor is a rotating wall vessel. In some embodiments, the bioreactor is a perfusion bioreactor. In some embodiments, the bioreactor is an isolation/expansion automated system. In some embodiments, the bioreactor is an automated or semi-automated bioreactor. In some embodiments, the bioreactor is a disposable bag bioreactor.

In some embodiments, the bioreactor has a volume of about 100 ml to about 1,000 liters. In some embodiments, the bioreactor has a volume of about 10 liters to about 1,000 liters. In some embodiments, the bioreactor has a volume of about 100 liters to about 900 liters. In some embodiments, the bioreactor has a volume of about 10 liters to about 800 liters. In some embodiments, the bioreactor comprises a volume of about 10 liters to about 700 liters, about 10 liters to about 600 liters, about 10 liters to about 500 liters, about 10 liters to about 400 liters, about 10 liters to about 300 liters, about 10 liters to about 200 liters, about 10 liters to about 100 liters, about 10 liters to about 90 liters, about 10 liters to about 80 liters, about 10 liters to about 70 liters, about 10 liters to about 60 liters, about 10 liters to about 50 liters, about 10 liters to about 40 liters, about 10 liters to about 30 liters, about 10 liters to about 20 liters, about 20 liters to about 1,000 liters, about 20 liters to about 900 liters, about 20 liters to about 800 liters, about 20 liters to about 700 liters, about 20 liters to about 600 liters, about 2 ... about 500 l, about 20 l to about 400 l, about 20 l to about 300 l, about 20 l to about 200 l, about 20 l to about 100 l, about 20 l to about 90 l, about 20 l to about 80 l, about 20 l to about 70 l, about 20 l to about 60 l, about 20 l to about 50 l, about 20 l to about 40 l, about 20 l to about 30 l, about 30 l to about 1,000 l, about 30 l to about 900 l, about 30 l to about 800 l, about 30 l to about 700 l, about 30 l to about 600 l, about 30 l to about 500 l, about 30 l to about 400 l, about 30 l to about 300 l, about 30 l to about 200 l, about 30 l to about 100 l, about 30 l to about 90 1, about 30l to about 80l, about 30l to about 70l, about 30l to about 60l, about 30l to about 50l, about 30l to about 40l, about 40l to about 1,000l, about 40l to about 900l, about 40l to about 800l, about 40l to about 700l, about 40l to about 600l, about 40l to about 500l, about 40l to about 400l, about 40l to about 300l, about 40l to about 200l, about 40l to about 100l, about 40l to about 90l, about 40l to about 80l, about 40l to about 70l, about 40l to about 60l, about 40l to about 50l, about 50l to about 1,000l, about 50l to about 900l, about 0l to about 800l, about 50l to about 700l, about 50l to about 600l, about 50l to about 500l, about 50l to about 400l, about 50l to about 300l, about 50l to about 200l, about 50l to about 100l, about 50l to about 90l, about 50l to about 80l, about 50l to about 70l, about 50l to about 60l, about 60l to about 1,000l, about 60l to about 900l, about 60l to about 800l, about 60l to about 700l, about 60l to about 600l, about 60l to about 500l, about 60l to about 400l, about 60l to about 300l, about 60l to about 200l, about 60l to about 100l, about 60 1 to about 90 1, about 60 1 to about 80 1, about 60 1 to about 70 1, about 70 1 to about 1,000 1, about 70 1 to about 900 1, about 70 1 to about 800 1, about 70 1 to about 700 1, about 70 1 to about 600 1, about 70 1 to about 500 1, about 70 1 to about 400 1, about 70 1 to about 300 1, about 70 1 to about 200 1, about 70 1 to about 100 1, about 70 1 to about 90 1, about 70 1 to about 80 1, about 80 1 to about 1,000 1, about 80 1 to about 900 1, about 80 1 to about 800 1, about 80 1 to about 700 1, about 80 1 to about 600 1, about 80 1 to about 500 1, about 80 1 to about 400 1, about 0L to about 300L, about 80L to about 200L, about 80L to about 100L, about 80L to about 90L, about 90L to about 1,000L, about 90L to about 900L, about 90L to about 800L, about 90L to about 700L, about 90L to about 600L, about 90L to about 500L, about 90L to about 400L, about 90L to about 300L, about 90L to about 200L, about 90L to about 100L, about 100L to about 1,000L, about 100L to about 900L, about 100L to about 800L, about 100L to about 700L, about 100L to about 600L, about 100L to about 500L, about 100L to about 400L, 100L to about 300L, about 100L to about 200L, about 200L to about 1,000L, about 200L to about 900L, about 200L to about 800L, about 200L to about 700L, about 200L to about 600L, about 200L to about 500L, about 200L to about 400L, about 200L to about 300L, about 300L to about 1,000L, about 300L to about 900L, about 300L to about 800L, about 300L to about 700L, about 300L to about 600L, about 300L to about 500L, about 300L to about 400L, about 400L to about 1,000L, about 400L to about 900L, about 400L to about 800 liters, about 400 liters to about 700 liters, about 400 liters to about 600 liters, about 400 liters to about 500 liters, about 500 liters to about 1,000 liters, about 500 liters to about 900 liters, about 500 liters to about 800 liters, about 500 liters to about 700 liters, about 500 liters to about 600 liters, about 600 liters to about 1,000 liters, about 600 liters to about 900 liters, about 600 liters to about 800 liters, about 600 liters to about 700 liters, about 700 liters to about 1,000 liters, about 700 liters to about 900 liters, about 700 liters to about 800 liters, about 800 liters to about 1,000 liters, about 800 liters to about 900 liters, or about 900 liters to about 1,000 liters. In some embodiments, the bioreactor has a volume of about 50 liters.

In some embodiments, the bioreactor has a volume of 100 ml to 1,000 l. In some embodiments, the bioreactor has a volume of 10 l to 1,000 l. In some embodiments, the bioreactor has a volume of 100 l to 900 l. In some embodiments, the bioreactor has a volume of 10 l to 800 l. In some embodiments, the bioreactor comprises a volume of 10-700 liters, 10-600 liters, 10-500 liters, 10-400 liters, 10-300 liters, 10-200 liters, 10-100 liters, 10-90 liters, 10-80 liters, 10-70 liters, 10-60 liters, 10-50 liters, 10-40 liters, 10-30 liters, 10-20 liters, 20-1,000 liters, 20-900 liters, 20-800 liters, 20-700 liters, 20-600 liters, 20-100 liters, Up to 500l, 20l to 400l, 20l to 300l, 20l to 200l, 20l to 100l, 20l to 90l, 20l to 80l, 20l to 70l, 20l to 60l, 20l to 50l, 20l to 40l, 20l to 30l, 30l to 1,000l, 30l to 900l, 30l to 800l, 30l to 700l, 30l to 600l, 30l to 500l, 30l to 400l, 30l to 300l, 30l to 200l, 30l to 100l, 30l to 90l, 30l-80l, 30l-70l, 30l-60l, 30l-50l, 30l-40l, 40l-1,000l, 40l-900l, 40l-800l, 40l-700l, 40l-600l, 40l-500l, 40l-400l, 40l-300l, 40l-200l, 40l-100l, 40l-90l, 40l-80l, 40l-70l, 40l-60l, 40l-50l, 50l-1,000l, 50l-900l, 50l-80 0l, 50l-700l, 50l-600l, 50l-500l, 50l-400l, 50l-300l, 50l-200l, 50l-100l, 50l-90l, 50l-80l, 50l-70l, 50l-60l, 60l-1,000l, 60l-900l, 60l-800l, 60l-700l, 60l-600l, 60l-500l, 60l-400l, 60l-300l, 60l-200l, 60l-100l, 60l-90l, 6 0l-80l, 60l-70l, 70l-1,000l, 70l-900l, 70l-800l, 70l-700l, 70l-600l, 70l-500l, 70l-400l, 70l-300l, 70l-200l, 70l-100l, 70l-90l, 70l-80l, 80l-1,000l, 80l-900l, 80l-800l, 80l-700l, 80l-600l, 80l-500l, 80l-400l, 80l-300l, 8 0l-200l, 80l-100l, 80l-90l, 90l-1,000l, 90l-900l, 90l-800l, 90l-700l, 90l-600l, 90l-500l, 90l-400l, 90l-300l, 90l-200l, 90l-100l, 100l-1,000l, 100l-900l, 100l-800l, 100l-700l, 100l-600l, 100l-500l, 100l-400l, 100l-300l , 100l-200l, 200l-1,000l, 200l-900l, 200l-800l, 200l-700l, 200l-600l, 200l-500l, 200l-400l, 200l-300l, 300l-1,000l, 300l-900l, 300l-800l, 300l-700l, 300l-600l, 300l-500l, 300l-400l, 400l-1,000l, 400l-900l, 400l-800l, 4 The bioreactor has a volume of 001 to 7001, 4001 to 6001, 4001 to 5001, 5001 to 1,0001, 5001 to 9001, 5001 to 8001, 5001 to 7001, 5001 to 6001, 6001 to 1,0001, 6001 to 9001, 6001 to 8001, 6001 to 7001, 7001 to 1,0001, 7001 to 9001, 7001 to 8001, 8001 to 1,0001, 8001 to 9001, or 9001 to 1,0001. In some embodiments, the bioreactor has a volume of 50 liters.

4. Cell Expansion and Stimulation In some embodiments, a natural killer cell source, for example, a single unit of umbilical cord blood, is co-cultured with feeder cells to produce expanded and stimulated NK cells.

In some embodiments, the co-culture is performed in a culture medium described herein, such as culture medium example #1 (Table 1) or culture medium example #2 (Table 2).

In some embodiments, the natural killer cell source, e.g., a cord blood single unit, comprises 1× ¹⁰ or about 1× ¹⁰ to 1× ¹⁰ or about 1× ¹⁰ total nucleated cells prior to amplification. In some embodiments, the natural killer cell source, e.g., a cord blood single unit, comprises 1× ¹⁰ or about 1× ¹⁰ to 1.5× ¹⁰ or about 1.5× ¹⁰ total nucleated cells prior to amplification. In some embodiments, the natural killer cell source, e.g., a cord blood single unit, comprises 1× ¹⁰ total nucleated cells prior to amplification. In some embodiments, the natural killer cell source, e.g., a cord blood single unit, comprises about 1×10 total nucleated cells prior to amplification. In some embodiments, the natural killer cell source, e.g., a cord blood single unit, comprises ¹ × ¹⁰ total nucleated cells prior to amplification. In some embodiments, a natural killer cell source, for example, a single unit of umbilical cord blood, contains approximately 1 x ¹⁰⁹ total nucleated cells prior to expansion.

In some embodiments, the natural killer cell source, e.g., cells derived from a co-culture of a single cord blood unit with feeder cells, are harvested and frozen, e.g., harvested and frozen in a cryopreservation composition as described herein. In some embodiments, the frozen cells derived from the co-culture are an infusion-ready drug product. In some embodiments, the frozen cells derived from the co-culture are used as a master cell bank (MCB) for producing an infusion-ready drug product, e.g., by one or more additional co-culture steps as described herein. Thus, for example, the natural killer cell source can be expanded and stimulated as described herein to produce expanded and stimulated NK cells suitable for use in an infusion-ready drug product without producing any intermediate product. The natural killer cell source can also be expanded and stimulated as described herein to produce an intermediate product, e.g., a primary master cell bank (MCB). The primary MCB can be used to produce expanded and stimulated NK cells suitable for use in an injectable-finished drug product, or alternatively, to produce other intermediate products, such as the secondary MCB. The secondary MCB can be used to produce expanded and stimulated NK cells suitable for use in an injectable-finished drug product, or alternatively, to produce other intermediate products, such as the tertiary MCB.

In some embodiments, the ratio of feeder cells:natural killer cell source or MCB cells inoculated into the co-culture is 1:1 or about 1:1 to 4:1 or about 4:1. In some embodiments, the ratio of feeder cells:natural killer cell source or MCB cells is 1:1 or about 1:1-3.5:1 or about 3.5:1, 1:1 or about 1:1-3:1 or about 3:1, 1:1 or about 1:1-2.5:1 or about 2.5:1, 1:1 or about 1:1-2:1 or about 2:1, 1:1 or about 1:1-1.5 or about 1.5:1, 1.5:1 or about 1.5:1-4:1 or about 4:1, 1.5:1 or about 1.5:1-3.5:1 or about 3.5:1, 1.5:1 or about 1.5:1-3:1 or about 3:1, 1.5:1 or about 1.5:1-2.5:1 or about 2.5:1, 2:1 or about 1.5:1 to 2:1 or about 2:1, 2:1 or about 2:1 to 4:1 or about 4:1, 2:1 or about 2:1 to 3.5:1 or about 3.5:1, 2:1 or about 2:1 to 3:1 or about 3:1, 2:1 or about 2:1 to 2.5:1 or about 2.5:1, 2.5:1 or about 2.5:1 to 4:1 or about 4:1, 2.5:1 or about 2.5:1 to 3.5:1 or about 3.5:1, 2.5:1 or about 2.5:1 to 3:1 or about 3:1, 3:1 or about 3:1 to 4:1 or about 4:1, 3:1 or about 3:1 to 3.5:1 or about 3.5:1, or 3.5:1 or about 3.5:1 to 4:1 or about 4:1. In some embodiments, the ratio of feeder cells to natural killer cell source or MCB cells inoculated into the co-culture is 2.5:1. In some embodiments, the ratio of feeder cells to natural killer cell source or MCB cells inoculated into the co-culture is about 2.5:1.

In some embodiments, the co-culture is performed in a disposable culture bag, e.g., a 1 L disposable culture bag. In some embodiments, the co-culture is performed in a bioreactor, e.g., a 50 L bioreactor. In some embodiments, culture medium is added to the co-culture after the primary inoculation.

In some embodiments, the co-culture is performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, or longer. In some embodiments, the co-culture is performed for up to 16 days.

In some embodiments, the co-culture is performed at or about 37°C.

In some embodiments, the co-culture is carried out at or about pH 7.9.

In some embodiments, the co-culture is carried out at a dissolved oxygen (DO) level of 50% or greater.

In some embodiments, exemplary culture medium #1 (Table 1) is used to produce MCB and culture medium #2 (Table 2) is used to produce cells suitable for injection-finished drug product.

In some embodiments, a natural killer cell source, such as a single unit of umbilical cord blood, is co-cultured with feeder cells to produce cells, such as MCB cells or infusion-ready drug product cells ^, at or about 50×10 ⁸ to 50×10 ¹² or about 50×10 ¹² . In some embodiments, such expansion produces cells, e.g., MCB cells or injectable-finished drug product cells, from ^50x10 or about 50x10 ^{to 25x10} or about 25x10 ^{, 10x10} or about 10x10 to ^1x10 or about ^{1x10 , 50x10 or about 50x10 to 75x10 or about 75x10, 50x10 or about 50x10 to 50x10 or} about ^50x10 , ^50x10 or about ^50x10 to ^25x10 or about 25x10, ^50x10 or about 50x10 ^{to 1x10} or about ^{1x10 , 50x10} or ^{about 50x10} up to ^75x108 or about ^75x108 , ^75x108 or about 75x108 to ^50x1010 or about ^{50x1010 , 75x108} or about 75x108 to ^25x1010 or about ^{25x1010 , 75x108 or about 75x108 to 1x1010 or about 1x1010 , 75x108 or} about 75x108 to ^75x109 or about ^75x109 , ^{75x108 or} about ^75x108 to ^50x109 or about ^50x109 , ^75x108 or about ^75x108 to ^25x109 or about ^25x109 , ^75x108 or about ^75x108 up to 1x10 ⁹ or about 1x10 ⁹ , 1x10 ⁹ or about 1x10 ⁹ to 50x10 ¹⁰ or about 50x10 10 , 1x10 ⁹ or about 1x10 9 to 25x10 ¹⁰ or about 25x10 ¹⁰ , 1x10 9 or about 1x10 ⁹ to 1x10 ¹⁰ or about ^{1x10 10 ,} 1x10 ⁹ or about 1x10 ⁹ to 75x10 ⁹ or about 75x10 9 , 1x10 ⁹ or about 1x10 ⁹ to 50x10 ⁹ or ^{about 50x10 9 , 1x10 9 or about 1x10 9 to 25x10 9 or about 25x10 9 , 25x10 9 or about 25x10 9 to 50x10 10} or about 50x10 ¹⁰ , ^25x109 or about 25x109 ^{to 25x1010} or about 25x1010, ^25x109 or about ^25x109 to ^1x1010 or about ^1x1010 , ^25x109 or about ^25x109 to ^75x109 or about ^75x109 , ^25x109 or about ^25x109 to ^{50x109 or} about 50x109, ^50x109 or about ^50x109 to ^50x1010 or about ^{50x1010, 50x109 or} about ^50x109 to ^25x1010 or about ^25x1010 , ^50x109 or about ^50x109 to ^1x1010 or about ^{1x10 10} , ^50x109 or about 50x109 to ^75x109 or about 75x109, ^75x109 or about 75x109 to ^50x1010 or about ^50x1010 , ^75x109 or about ^75x109 to ^25x1010 or about ^25x1010 , ^75x109 or about ^{75x109 to 1x1010} or about ^1x1010 , ^1x1010 or about ^1x1010 to ^50x1010 or about 50x1010, ^{1x1010 or about 1x1010} to ^25x1010 or about ^25x1010 , or ^25x1010 or about ^25x1010 to ^{50x10 10} or approximately 50×10 ¹⁰ are produced.

In some embodiments, the amplification produces 60 or about 60-100 or about 100 vials containing 600 million or about 600 million to 1 billion or about 1 billion cells, e.g., MCB cells or injectable-finished drug product cells, respectively. In some embodiments, the amplification produces 80 or about 80 vials containing or consisting of 800 million or about 800 million cells, e.g., MCB cells or injectable-finished drug product cells, respectively.

In some embodiments, the expansion increases the number of cells, e.g., MCB cells, by 100-fold or about 100-fold to 500-fold or about 500-fold relative to the number of cells, e.g., NK cells, in the natural killer cell source. In some embodiments, the expansion is by increasing the number of cells, e.g., the number of cells of MCB cells, relative to the number of cells in a natural killer cell source, e.g., the number of cells of NK cells, by 100-fold or about 100-fold to 500-fold or about 500-fold, 100-fold or about 100-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 300-fold or about 300-fold, 100-fold or about 100-fold to 200-fold or about 200-fold, 200-fold or about 200-fold to 450-fold or about 450-fold, 200-fold or about 200-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 350-fold or about 350-fold, 200-fold or about 200-fold to 300-fold or about 300-fold, 200-fold or about 200-fold to 250-fold or about 250-fold, 250-fold or about 200-fold to 300-fold. 50-500-fold or about 500-fold, 250-fold or about 250-fold or about 450-fold, 200-fold or about 200-fold or about 400-fold, 250-fold or about 250-fold or about 350-fold, 250-fold or about 250-fold or about 300-fold, 300-fold or about 300-fold or about 500-fold, 300-fold or about 3 00-450-fold or about 450-fold, 300-fold or about 300-fold or about 400-fold, 300-fold or about 300-fold or about 350-fold, 350-fold or about 350-fold or about 500-fold, 350-fold or about 350-fold or about 450-fold, or 350-fold or about 350-fold or about 400-fold.

In some embodiments, the amplification increases the number of cells, e.g., MCB cells, by 100-fold or about 100-fold to 70,000-fold or about 70,000-fold relative to the number of cells, e.g., NK cells, in the natural killer cell source. In some embodiments, the amplification increases the number of cells, e.g., MCB cells, by at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold, or 70,000-fold relative to the number of cells, e.g., NK cells, in the natural killer cell source.

In some embodiments, co-culture of MCB cells with feeder cells produces cells, e.g., 500 million or about 500 million to 1.5 billion or about 1.5 billion NK cells suitable for use in the MCB and/or injectable finished pharmaceutical product. In some embodiments, co-culture of MCB cells with feeder cells produces cells, e.g., 500 million or about 500 million to 1.5 billion or about 1.5 billion, 500 million or about 500 million to 1.25 billion or about 1.25 billion, 500 million or about 500 million to 1 billion or about 1 billion, 500 million or about 500 million to 750 million or about 750 million, 750 million or about 750 million to 1.5 billion or about 1.5 billion, 500 million or about 500 million to 1.25 billion or about 1.25 billion, 750 million or about 750 million to 1 billion or about 1 billion, 1 billion or about 1 billion to 1.5 billion or about 1.5 billion, 1 billion or about 1 billion to 1.25 billion or about 1.25 billion, or 1.25 billion to 1.5 billion or about 1.5 billion NK cells suitable for use in MCB and/or injectable finished pharmaceutical products.

In some embodiments, the co-culture of MCB cells with feeder cells produces 50 or about 50 to 150 or about 150 vials of cells, e.g., injectable-finished drug product cells, containing 750 or about 750 to 1.25 billion or about 1.25 billion MCB and/or NK cells suitable for use in an injectable-finished drug product, respectively. In some embodiments, the co-culture of MCB cells with feeder cells produces 100 or about 100 vials containing or consisting of 1 billion or about 1 billion MCB and/or NK cells suitable for use in an injectable-finished drug product, respectively.

In some embodiments, the expansion increases the number of cells suitable for use in the MCB and/or injectable-finished pharmaceutical product, e.g., NK cell numbers, by 100-fold or about 100-fold to 500-fold or about 500-fold compared to the initial MCB cell numbers. In some embodiments, the expansion involves increasing the cell numbers, e.g., increasing the cell numbers of the MCB and/or NK cells suitable for use in an injectable-finished drug product by 100-fold or about 100-fold to 500-fold or about 500-fold, 100-fold or about 100-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 300-fold or about 300-fold, 100-fold or about 100-fold to 200-fold or about 200-fold, 200-fold or about 200-fold to 450-fold or about 450-fold, 200-fold or about 200-fold to 400-fold or about 400-fold, 100-fold or about 100-fold to 350-fold or about 350-fold, 200-fold or about 200-fold to 300-fold or about 300-fold, 200-fold or about 200-fold to 250-fold or about 250-fold, 250-fold or about 250-fold to 500-fold or about 500-fold, 250-fold or about 250-fold to 450-fold or about 450-fold, 200-fold or about 200-fold to 400-fold or about 400-fold, 250-fold or about 250-fold to 350-fold or about 350-fold, 250-fold or about 250-fold to 300-fold or about 300-fold, 300-fold or about 300-fold to 500-fold or about 500-fold, up to 300-fold or about 300-fold to 450-fold or about 450-fold, 300-fold or about 300-fold to 400-fold or about 400-fold, 300-fold or about 300-fold to 350-fold or about 350-fold, 350-fold or about 350-fold to 500-fold or about 500-fold, 350-fold or about 350-fold to 450-fold or about 450-fold, 350-fold or about 350-fold to 400-fold or about 400-fold.

In embodiments in which cells are engineered during expansion and stimulation as described herein, not all expanded and stimulated cells will necessarily be successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising CAR and/or IL-15, as described herein. Thus, the methods described herein can additionally include sorting the engineered cells, e.g., the engineered cells described herein, from non-engineered cells.

In some embodiments, the engineered cells, e.g., transduced cells, are separated from non-engineered cells, e.g., non-transduced cells, using a reagent specific for an antigen of the engineered cells, e.g., an antibody that targets an antigen of the engineered cells but not the non-engineered cells. In some embodiments, the antigen of the engineered cells is a component of a CAR, e.g., a component of a CAR described herein.

Systems for antigen-based cell separation are commercially available, for example the ^CliniMAC® sorting system (Miltenyi Biotec).

In some embodiments, engineered cells, e.g., transduced cells, are separated from non-engineered cells, e.g., non-transduced cells, using flow cytometry measurements.

In some embodiments, the sorted engineered cells are used as MCBs. In some embodiments, the sorted engineered cells are used as components of injectable-finished pharmaceutical products.

In some embodiments, engineered cells, e.g., transduced cells, are sorted from non-engineered cells, e.g., non-transduced cells, using microfluidic cell sorting methods. Microfluidic cell sorting methods are described, for example, in Dalili et al., "A Review of Sorting, Separation and Isolation of Cells and Microbeads for Biomedical Applications: Microfluidic Approaches," Analyst 144:87 (2019).

In some embodiments, 1% or about 1% to 99% or about 99% of the expanded and stimulated cells are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising CAR and/or IL-15, as described herein. In some embodiments, 1% or about 1% to 90% or about 90%, 1% or about 1% to 80% or about 80%, 1% or about 1% to 70% or about 70%, 1% or about 1% to 60% or about 60%, 1% or about 1% to 50% or about 50%, 1% or about 1% to 40% or about 40%, 1% or about 1% to 30% or about 30%, 1% or about 1% to 20% or about 20%, 1% or about 1% of the expanded and stimulated cells are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising CAR and/or IL-15, as described herein. % to 10% or about 10%, 1% or about 1% to 5% or about 5%, 5% or about 5% to 99% or about 99%, 5% or about 5% to 90% or about 90%, 5% or about 5% to 80% or about 80%, 5% or about 5% to 70% or about 70%, 5% or about 5% to 60% or about 60%, 5% or about 5% to 50% or about 50%, 5% or about 5% to 40% or about 40%, 5% or about 5% to 30% or about 30%, 5% or about 5% to 50% or about 50%, % or about 5% to 20% or about 20%, 5% or about 5% to 10% or about 10%, 10% or about 10% to 99% or about 99%, 10% or about 10% to 90% or about 90%, 10% or about 10% to 80% or about 80%, 10% or about 10% to 70% or about 70%, 10% or about 10% to 60% or about 60%, 10% or about 10% to 50% or about 50%, 10% or about 10% to 40% or about 40% , 10% or about 10% to 30% or about 30%, 10% or about 10% to 20% or about 20%, 20% or about 20% to 99% or about 99%, 20% or about 20% to 90% or about 90%, 20% or about 20% to 80% or about 80%, 20% or about 20% to 70% or about 70%, 20% or about 20% to 60% or about 60%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 40% or about 40%, 20% or about 20% to 30% or about 30%, 30% or about 30% to 99% or about 99%, 30% or about 30% to 90% or about 90%, 30% or about 30% to 80% or about 80%, 30% or about 30% to 70% or about 70%, 30% or about 30% to 60% or about 60%, 30% or about 30% to 50% or about 50%, 30% or about 30% to 40% or about 40%, 40% or about 40 % to 99% or about 99%, 40% or about 40% to 90% or about 90%, 40% or about 40% to 80% or about 80%, 40% or about 40% to 70% or about 70%, 40% or about 40% to 70% or about 70%, 40% or about 40% to 60% or about 60%, 40% or about 40% to 50% or about 50%, 50% or about 50% to 99% or about 99%, 50% or about 50% to 90% or about 90%, 50% or about 50% to 80% or about 80%, 50% or about 50% to 70% or about 70%, 50% or about 50% to 60% or about 60%, 60% or about 60% to 99% or about 99%, 60% or about 60% to 90% or about 90%, 60% or about 60% to 80% or about 80%, 60% or about 60% to 70% or about 70%, 70% or about 70% to 99% or about 99%, 70% or about 70% to 90% or about 9 0%, 70% or about 70%-80% or about 80%, 80% or about 80%-99% or about 99%, 80% or about 80%-90% or about 90%, or 90% or about 90%-99% or about 99% are successfully engineered, e.g., successfully transduced, e.g., successfully transduced with a vector comprising a heterologous protein, e.g., a heterologous protein comprising CAR and/or IL-15, as described herein.

In some embodiments, frozen cells of the primary or secondary MCB are thawed and cultured. In some embodiments, a single vial of frozen cells of the primary or secondary MCB, e.g., a single vial containing at or about 800 million cells of the primary or secondary MCB cells, is thawed and cultured. In some embodiments, the frozen primary or secondary MCB cells are cultured with additional feeder cells to produce cells suitable for use as a secondary or tertiary MCB or for injectable-finished drug products. In some embodiments, cells are harvested and frozen from the co-culture of the primary or secondary MCB.

In some embodiments, cells are harvested from a co-culture of a natural killer cell source, primary MCB, or secondary MCB, and frozen in a cryopreservation composition, e.g., a cryopreservation composition described herein. In some embodiments, the cells are washed after harvesting. Thus, the present invention provides pharmaceutical compositions and cryopreservation compositions, e.g., a cryopreservation composition described herein, comprising activated and stimulated NK cells, e.g., activated and stimulated NK cells produced by the methods described herein, e.g., harvested and washed activated and stimulated NK cells produced by the methods described herein.

In some embodiments, the cells are mixed with a cryopreservation composition prior to freezing, e.g., as described herein. In some embodiments, the cells are frozen in a cryobag. In some embodiments, the cells are frozen in a cryovial.

In some embodiments, the method additionally includes isolating NK cells from the expanded and stimulated population of NK cells.

An example of a process for expanding and stimulating NK cells is shown in Figure 1.

5. Engineering In some embodiments, the method additionally includes engineering the NK cells to express a heterologous protein, e.g., a heterologous protein, including a heterologous protein described herein, e.g., a CAR and/or IL-15.

In some embodiments, engineering the NK cells to express a heterologous protein described herein comprises transforming, e.g., stably transforming, the NK cells with a vector comprising a polynucleic acid encoding a heterologous protein described herein. Suitable vectors are described herein.

In some embodiments, engineering NK cells to express a heterologous protein as described herein includes introducing the heterologous protein via gene editing (e.g., zinc finger nuclease (ZFN) gene editing, ARCUS gene editing, CRISPR-Cas9 gene editing, or megaTAL gene editing) in combination with adeno-associated virus (AAV) technology.

In some embodiments, the NK cells are engineered to express a heterologous protein described herein, e.g., during or after culturing the composition in medium containing feeder cells.

In some embodiments, the method additionally includes engineering the NK cells, e.g., to express, overexpress, knock out, or knock down a gene or gene product.

In some embodiments, the natural killer cells are not genetically engineered.

E. Properties of Expanded and Stimulated NK Cells After ex vivo expansion and stimulation, for example as described herein, the expanded and stimulated NK cell population not only has a number/density (e.g., as described above) that does not naturally occur in the human body, but also has phenotypic characteristics (e.g., gene expression and/or surface protein expression) that differ from the starting source or other naturally occurring populations of NK cells.

In some cases, the starting NK cell source is a sample derived from a single individual, e.g., a single umbilical cord blood unit without ex vivo expansion. Thus, in some cases, the expanded and stimulated NK cells share a common lineage, i.e., they are all generated from the expansion of the starting NK cell source, and thus share a genotype due to the clonal expansion of a cell population that is itself derived from a single organism. However, these cells may not naturally occur at the densities achieved by ex vivo expansion, and differences in phenotypic characteristics exist from the starting NK cell source.

In some cases, the population of expanded and stimulated NK cells includes at least 100 million, e.g., 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 90 billion, 100 billion, 200 billion, 250 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion expanded natural killer cells.

In some embodiments, the expanded and stimulated NK cells comprise at least 80%, e.g., at least 90%, at least 95%, at least 99% or 100% CD56+CD3- cells.

In some embodiments, the expanded and stimulated NK cells are not genetically engineered.

In some embodiments, the expanded and stimulated NK cells do not contain a CD16 transgene.

In some embodiments, the expanded and stimulated NK cells do not express exogenous CD16 protein.

Expanded and stimulated NK cells are characterized, for example, by surface expression of one or more of CD16, CD56, CD3, CD38, CD14, CD19, NKG2D, NKp46, NKp30, DNAM-1, and NKp44.

The expression levels of the surface proteins referenced herein are, in some cases, achieved without positive selection for the particular proteins referenced. For example, in some cases, an NK cell source, e.g., a single cord blood unit, contains both the KIR B allele of the KIR receptor system and the 158V/V variant of CD16, and is CD56+ enriched and CD3(+) depleted, e.g., by gating on CD56+CD3- expression, but does not select for other surface protein expression during expansion and stimulation.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKG2D+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp46+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp30+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% DNAM-1+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp44+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, comprise at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% CD94+(KLRD1) cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD3+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD14+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD19+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CXCR+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, contain 20% or less, e.g., 10% or less, 5% or less, 1% or less, or 0% CD122+ (IL2RB) cells.

As described herein, the inventors have surprisingly found that NK cells expanded and stimulated by the methods described herein express high levels of CD16 due to the expansion and stimulation process, resulting in a cell population with high CD16 expression. The high expression of CD16 obviates the need to engineer the expanded cells to express CD16, which is important for initiating ADCC, which is a surprising and unexpected advantage of the expansion and stimulation methods described herein. Thus, in some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, as described above, contain 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells.

In some embodiments, the expanded and stimulated NK cells, e.g., as described above, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, contain both the KIR B allele of the KIR receptor system and the 158V/V variant of CD16, and comprise 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells.

In some embodiments, the percentage of expanded and stimulated NK cells expressing CD16, e.g., as described above, derived from the expansion and stimulation of a single cord blood unit, is the same as or higher than the percentage of natural killer cells in the seed cells derived from the cord blood.

In some embodiments, the percentage of expanded and stimulated NK cells that express NKG2D, e.g., as described above, derived from the expansion and stimulation of a single cord blood unit, is the same as or higher than the percentage of natural killer cells in the seed cells derived from the cord blood.

In some embodiments, the percentage of expanded and stimulated NK cells that express NKp30, e.g., as described above, derived from the expansion and stimulation of a single cord blood unit, is the same as or higher than the percentage of natural killer cells in the seed cells derived from the cord blood.

In some embodiments, the percentage of expanded and stimulated NK cells that express DNAM-1, e.g., as described above, derived from the expansion and stimulation of a single cord blood unit, is the same as or higher than the percentage of natural killer cells in the seed cells derived from the cord blood.

In some embodiments, the percentage of expanded and stimulated NK cells that express NKp44, e.g., as described above, derived from the expansion and stimulation of a single cord blood unit, is the same as or higher than the percentage of natural killer cells in the seed cells derived from the cord blood.

In some embodiments, the percentage of expanded and stimulated NK cells that express NKp46, e.g., as described above, derived from the expansion and stimulation of a single cord blood unit, is the same as or higher than the percentage of natural killer cells in the seed cells derived from the cord blood.

As described herein, the inventors have surprisingly found that NK cells expanded and stimulated by the methods described herein express low levels of CD38. CD38 is an effective target in certain cancer therapies (e.g., multiple myeloma and acute myeloid leukemia). See, e.g., Jiao et al., "CD38: Targeted Therapy in Multiple Myeloma and Therapeutic Potential for Solid Cancers," Expert Opinion on Investigational Drugs 29(11):1295-1308 (2020). However, when anti-CD38 antibodies are administered together with NK cells, there is a risk of increased fratricide since NK cells naturally express CD38. However, NK cells expanded and stimulated by the methods described herein express low levels of CD38, thus eliminating the expected fratricide. Other groups have relied on engineering methods such as genome editing to reduce CD38 expression (e.g., Gurney et al., "CD38 Knockout Natural Killer Cells Expressing an Affinity Optimized CD38 Chimeric Antigen Receptor Successfully Target Acute Myoid Leukemia with Reduced Effector Cell Fragrance," Haematologica, 2011). doi:10.3324/haematol.2020.271908(2020)), NK cells expanded and stimulated by the methods described herein express low levels of CD38 without genetic manipulation, providing surprising and unexpected advantages for treating, for example, CD38+ cancers using NK cells expanded and stimulated as described herein, e.g., in combination with a CD38 antibody.

Thus, in some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, as described above, comprise 80% or less CD38+ cells, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less CD38+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., as described above, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, contain both the KIR B allele of the KIR receptor system and the 158V/V variant of CD16, and contain 80% or less CD38+ cells, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less CD38+ cells.

In some embodiments, the expanded and stimulated NK cells, e.g., as described above, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, comprise both the KIR B allele of the KIR receptor system and the 158V/V variant of CD16, and comprise 80% or less, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, or 20% or less, CD38+ cells, and 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells.

In some embodiments, the expanded and stimulated NK cells, e.g., expanded and stimulated NK cells derived from the expansion and stimulation of a single cord blood unit, e.g., as described above, contain both the KIR B allele of the KIR receptor system and the 158V/V variant of CD16, and include: i) 50% or more, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% CD16+ NK cells; and/or ii) 80% or less CD38+ cells, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less or 20% or less CD38+ cells; and/or iii) at least 60%, e.g., at least 70%, at least and/or iv) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of NKG2D+ cells; and/or iv) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of NKp46+ cells; and/or v) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of NKp30+ cells; and/or vi) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of NKp46+ cells; and/or vii) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of NKp44+ cells; and/or viii) at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of CD94+ (KLRD1) cells; and/or ix) 2 0% or less, e.g., 10% or less, 5% or less, 1% or less or 0% of CD3+ cells; and/or x) 20% or less, e.g., 10% or less, 5% or less, 1% or less or 0% of CD14+ cells; and/or xi) 20% or less, e.g., 10% or less, 5% or less, 1% or less or 0% of CD19+ cells; and/or xii) 20% or less, e.g., 10% or less, 5% or less, 1% or less or 0% of CXCR+ cells; and/or xiii) 20% or less, e.g., 10% or less, 5% or less, 1% or less or 0% of CD122+ (IL2RB) cells.

In some embodiments, a residual signature of the feeder cells may be detected, for example, by the presence of residual cells (e.g., by detecting cells expressing a particular surface protein) or by residual nucleic acids and/or proteins expressed by the feeder cells, but which do not persist in the NK cells that were expanded and stimulated by the feeder cells.

For example, in some cases, the methods described herein include expanding and stimulating natural killer cells using engineered feeder cells, e.g., the eHuT-78 feeder cells described above, engineered to express sequences not expressed by cells in a natural killer cell source, such as natural killer cells. For example, the engineered feeder cells can be engineered to express one or more genes selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2), and mutant TNFα (SEQ ID NO:3) ("eHut-78 cells"), or variants thereof.

While these feeder cells may not persist in the expanded and stimulated NK cells, the expanded and stimulated NK cells may retain detectable residual amounts of cells, proteins and/or nucleic acids derived from the feeder cells. Thus, the presence of such residues in the expanded and stimulated NK cells can be detected, for example, by detecting the cells themselves (e.g., by flow cytometric measurements), proteins expressed by these cells, and/or nucleic acids expressed by these cells.

To this end, the present invention also describes a population of expanded and stimulated NK cells that contain residual feeder cells (viable or dead) or cellular impurities of the residual feeder cells (e.g., proteins or parts thereof, and/or genetic material such as nucleic acids or parts thereof of the residual feeder cells). In some cases, the expanded and stimulated NK cells contain residual feeder cells, e.g., eHuT-78 feeder cells, at a level greater than 0% and less than or equal to 0.3%.

In some cases, the expanded and stimulated NK cells contain residual feeder cells encoding, for example, residual 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2), and/or mutant TNFα (SEQ ID NO:3), or portions thereof. In some cases, the membrane-bound IL-21 contains the CD8 transmembrane domain.

In some cases, the expanded and stimulated NK cells contain a % residual feeder cell of greater than 0% and less than or equal to 0.2%, e.g., as measured by the relative proportion of feeder cell-specific proteins or nucleic acid sequences (i.e., proteins or nucleic acid sequences not expressed by natural killer cells) in the sample, e.g., by qPCR, e.g., as described herein.

In some embodiments, the remaining feeder cells are CD4(+) T cells. In some embodiments, the remaining feeder cells are engineered CD4(+) T cells. In some embodiments, the remaining feeder cells are engineered to express one or more genes selected from the group consisting of 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2) and mutant TNFα (SEQ ID NO:3) ("eHut-78 cells"), or variants thereof. Thus, in some cases, the feeder cell specific protein is 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2) and/or mutant TNFα (SEQ ID NO:3). Thus, the feeder cell-specific nucleic acid is a nucleic acid encoding 4-1BBL (UniProtKB P41273, SEQ ID NO:1), membrane-bound IL-21 (SEQ ID NO:2) and/or mutant TNFα (SEQ ID NO:3), or a portion thereof. In some cases, the membrane-bound IL-21 comprises the CD8 transmembrane domain.

A wide variety of different methods can be used to analyze and detect the presence of nucleic acid or protein gene products in biological samples. As used herein, "detection" can refer to a method used to discover, confirm or verify the coexistence or presence of a compound and/or substance (e.g., a cell, a protein and/or a nucleic acid). In some embodiments, a detection method can be used to detect a protein. In some embodiments, detection can include chemiluminescent or fluorescent techniques. In some embodiments, detection can include immunologically-based methods (e.g., quantitative enzyme-linked immunosorbent assay (ELISA), Western blotting or dot blotting) that use antibodies to specifically react with the whole protein or specific epitopes of the protein. In some embodiments, detection can include immunoprecipitation of the protein (Jungblut et al., J Biotechnol. 31;41(2-3):111-20 (1995); Franco et al., Eur J Morphol. 39(1):3-25 (2001)). In some embodiments, detection methods can be used to detect nucleic acids (e.g., DNA and/or RNA). In some embodiments, detection can include Northern blot analysis, nuclease protection assay (NPA), in situ hybridization, or reverse transcription-polymerase chain reaction (RT-PCR) (Raj et al., Nat. Methods 5, 877-879 (2008); Jin et al., J Clin Lab Anal. 11(1):2-9 (1997); Ahmed, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 20(2):77-116 (2002)).

To this end, the present invention also describes a method for detecting a population of expanded and stimulated NK cells co-cultured with engineered feeder cells, e.g., eHuT-78 feeder cells, as described herein, e.g., a population of expanded and stimulated NK cells using the methods described herein.

II. Cryopreservation A. Cryopreservation Compositions The present invention provides cryopreservation compositions, e.g., cryopreservation compositions suitable for intravenous administration, e.g., for intravenous administration of NK cells, e.g., NK cells described herein. In some embodiments, a pharmaceutical composition comprises the cryopreservation composition and cells, e.g., NK cells described herein.

1. Albumin In some embodiments, the cryopreservation composition comprises an albumin protein, e.g., human albumin protein (UniProtKB Accession P0278, SEQ ID NO:5), or a variant thereof. In some embodiments, the cryopreservation composition comprises an albumin protein, e.g., an ortholog of human albumin protein, or a variant thereof. In some embodiments, the cryopreservation composition comprises an albumin protein, e.g., a biologically active portion of human albumin, or a variant thereof.

In some embodiments, the albumin, e.g., human albumin, is provided as a solution, which is also referred to herein as an albumin solution or a human albumin solution. Thus, in some embodiments, the cryopreservation composition is or comprises an albumin solution, e.g., a human albumin solution. In some embodiments, the albumin solution is a serum-free albumin solution.

In some embodiments, the albumin solution is suitable for intravenous use.

In some embodiments, the albumin solution comprises 40 or about 40 to 200 or about 200 g/l of albumin. In some embodiments, the albumin solution comprises 40 or about 40 to 50 or about 50 g/l of albumin, e.g., human albumin. In some embodiments, the albumin solution comprises about 200 g/l of albumin, e.g., human albumin. In some embodiments, the albumin solution comprises 200 g/l of albumin, e.g., human albumin.

In some embodiments, the albumin solution comprises a protein composition that is 95% or more albumin protein, e.g., human albumin protein. In some embodiments, 96%, 97%, 98% or 99% or more of the protein is albumin, e.g., human albumin.

In some embodiments, the albumin solution additionally comprises sodium. In some embodiments, the albumin solution comprises 100 or about 100 to 200 or about 200 mmol of sodium. In some embodiments, the albumin solution comprises 130 or about 130 to 160 or about 160 mmol of sodium.

In some embodiments, the albumin solution additionally comprises potassium. In some embodiments, the albumin solution additionally comprises 3 mmol or less of potassium. In some embodiments, the albumin solution additionally comprises 2 mmol or less of potassium.

In some embodiments, the albumin solution additionally comprises one or more stabilizers. In some embodiments, the stabilizers are selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: acetyltryptophan, N-acetyl-L-tryptophan, and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: N-acetyltryptophan, DL-acetyltryptophan, and N-acetyl-DL-tryptophan). In some embodiments, the solution comprises less than 0.1 mmol of each of the one or more stabilizers per gram of protein in the solution. In some embodiments, the solution comprises 0.05 or about 0.05 to 0.1 or about 0.1, e.g., 0.064 or about 0.064 to 0.096 or about 0.096 mmol of each of the stabilizers per gram of protein in the solution. In some embodiments, the solution comprises less than 0.1 mmol of total stabilizer per gram of protein in the solution. In some embodiments, the solution comprises 0.05 or about 0.05 to 0.1 or about 0.1, e.g., 0.064 or about 0.064 to 0.096 or about 0.096 mmol of total stabilizer per gram of protein in the solution.

In some embodiments, the albumin solution is comprised of a protein composition of 95% or more albumin protein in water, one or more stabilizers selected from the group consisting of sodium, potassium and sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: acetyltryptophan, N-acetyl-L-tryptophan and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: N-acetyltryptophan, DL-acetyltryptophan and N-acetyl-DL-tryptophan).

In some embodiments, the cryopreservation composition comprises an albumin solution, such as an albumin solution described herein, at or about 10% v/v to 50% or about 50% v/v. In some embodiments, the cryopreservation composition comprises an albumin solution, such as an albumin solution described herein, at or about 10% v/v to 50% or about 50%, at or about 10% v/v to 45% or about 45%, at or about 10% v/v to 40% or about 40%, at or about 10% v/v to 35% or about 35%, at or about 10% v/v to 30% or about 30%, at or about 10% v/v to 25% or about 25%, at or about 10% v/v to 20% or about 20%, at or about 10% v/v to 15% or about 15%, at or about 15% v/v to 50% or about 50%, ...50% or about 50%, at or about 10% v/v to 45 0% or about 50%, 15% or about 15% to 45% or about 45%, 15% or about 15% to 40% or about 40%, 15% or about 15% to 35% or about 35%, 15% or about 15% to 30% or about 30%, 15% or about 15% to 25% or about 25%, 15% or about 15% to 20% or about 20%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 45% or about 45%, 20% or about 20% to 40% or about 40%, 0% or about 20% to 35% or about 35%, 20% or about 20% to 30% or about 30%, 20% or about 20% to 25% or about 25%, 25% or about 25% to 50% or about 50%, 25% or about 25% to 45% or about 45%, 25% or about 25% to 40% or about 40%, 25% or about 25% to 35% or about 35%, 25% or about 25% to 30% or about 30%, 30% or about 30% to 50% or about 50%, 30% or about 30% to 4 In some embodiments, the cryopreservation composition comprises an albumin solution described herein at about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v/v. In some embodiments, the cryopreservation composition comprises an albumin solution described herein at 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% v/v.

In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 20 or about 20-100 or about 100 g/l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 20 or about 20-100 or about 100, 20 or about 20-90 or about 90, 20 or about 20-80 or about 80, 20 or about 20-70 or about 70, 20 or about 20-60 or about 60, 20 or about 20-50 or about 50, 20 or about 20-40 or about 40, 20 or about 20-30 or about 30, 30 or about 30-10 0 or about 100, 30 or about 30-90 or about 90, 30 or about 30-80 or about 80, 30 or about 30-70 or about 70, 30 or about 30-60 or about 60, 30 or about 30-50 or about 50, 30 or about 30-40 or about 40, 40 or about 40-100 or about 100, 40 or about 40-90 or about 90, 40 or about 40-80 or about 80, 40 or about 40-70 or about 70, 40 or about 40-60 or about 60, 40 or about 40-50 or about 50, 50 or about 50-100 or about 100, 50 or about 50-90 or about 90, 50 or about 50-80 or about 80, 50 or about 50-70 or about 70, 50 or about 50-60 or about 60, 60 or about 60-100 or about 100, 60 or about 60-90 or about 90 , 60 or about 60-80 or about 80, 60 or about 60-70 or about 70, 70 or about 70-100 or about 100, 70 or about 70-90 or about 90, 70 or about 70-80 or about 80, 80 or about 80-100 or about 100, 80 or about 80-90 or about 90, or 90 or about 90-100 or about 100 g/l of albumin, e.g., human albumin.

In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 20 g/l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 40 g/l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 70 g/l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at 100 g/l.

In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 20 g/l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 40 g/l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 70 g/l. In some embodiments, the cryopreservation composition comprises albumin, e.g., human albumin, at about 100 g/l.

In some embodiments, the cryopreservation composition additionally comprises a stabilizer, e.g., an albumin stabilizer. In some embodiments, the stabilizer is selected from the group consisting of sodium caprylate, caprylic acid, (2S)-2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: acetyltryptophan, N-acetyl-L-tryptophan, and acetyl-L-tryptophan), 2-acetamido-3-(1H-indol-3-yl)propanoic acid (also referred to herein as: N-acetyltryptophan, DL-acetyltryptophan, and N-acetyl-DL-tryptophan). In some embodiments, the cryopreservation composition comprises less than 0.1 mmol of each of one or more stabilizers per gram of protein, e.g., albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises 0.05 or about 0.05 to 0.1 or about 0.1, e.g., 0.064 or about 0.064 to 0.096 or about 0.096 mmol of stabilizer per gram of protein, e.g., albumin protein, in the composition. In some embodiments, the cryopreservation composition comprises less than 0.1 mmol of total stabilizer per gram of protein, e.g., albumin protein, in the cryopreservation composition. In some embodiments, the cryopreservation composition comprises 0.05 or about 0.05 to 0.1 or about 0.1, e.g., 0.064 or about 0.064 to 0.096 or about 0.096 mmol of total stabilizer per gram of protein, e.g., albumin protein, in the cryopreservation composition.

2. Dextran In some embodiments, the cryopreservation composition comprises dextran or a derivative thereof.

Dextran is an anhydroglucose polymer (designated (C ₆ H ₁₀ O ₅ ) _n ) composed of approximately 95% α-D-(1-6) linkages. Dextran fractions are available in molecular weights ranging from about 1,000 Daltons to about 2,000,000 Daltons. This is designated by a number (Dextran X), e.g., Dextran 1, Dextran 10, Dextran 40, Dextran 70, etc., where X is the average molecular weight divided by 1,000 Daltons. Thus, for example, Dextran 40 has an average molecular weight of at or about 40,000 Daltons.

In some embodiments, the average molecular weight of the dextran is at or about 1,000 Daltons to 2,000 or about 2,000,000 Daltons. In some embodiments, the average molecular weight of the dextran is at or about 40,000 Daltons. In some embodiments, the average molecular weight of the dextran is at or about 70,000 Daltons.

In some embodiments, the dextran is selected from the group consisting of dextran 40, dextran 70, and combinations thereof. In some embodiments, the dextran is dextran 40.

In some embodiments, the dextran, e.g., dextran 40, is provided in solution, and as referred to herein, as a dextran solution or as a dextran 40 solution. Thus, in some embodiments, the composition comprises a dextran solution, e.g., a dextran 40 solution.

In some embodiments, the dextran solution is suitable for intravenous use.

In some embodiments, the dextran solution comprises about 5% to about 50% w/w of dextran, e.g., dextran 40. In some embodiments, the dextran solution comprises 5% or about 5% to 50% or about 50%, 5% or about 5% to 45% or about 45%, 5% or about 5% to 40% or about 40%, 5% or about 5% to 35% or about 35%, 5% or about 5% to 30% or about 30%, 5% or about 5% to 25% or about 25%, 5% or about 5% to 20% or about 20%, 5% or about 5% to 15 or about 15%, 5% or about 5% to 10% or about 10%, 10% or about 10% to 50% or about 50%, 10% or about 10% to 45% or about 45%, 10% or about 10% up to 40% or about 40%, 10% or about 10% to 35% or about 35%, 10% or about 10% to 30% or about 30%, 10% or about 10% to 25% or about 25%, 10% or about 10% to 20% or about 20%, 10% or about 10% to 15 or about 15%, 15% or about 15% to 50% or about 50%, 15% or about 15% to 45% or about 45%, 15% or about 15% to 40% or about 40%, 15% or about 15% to 35% or about 35%, 15% or about 15% to 30% or about 30%, 15% or about 15% to 25% or about 25%, 15% % or about 15% to 20% or about 20%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 45% or about 45%, 20% or about 20% to 40% or about 40%, 20% or about 20% to 35% or about 35%, 20% or about 20% to 30% or about 30%, 20% or about 20% to 25% or about 25%, 25% or about 25% to 50% or about 50%, 25% or about 25% to 45% or about 45%, 25% or about 25% to 40% or about 40%, 25% or about 25% to 35% or about 35%, 25% or about 25% to 30% or about 30%, 30% or about 30%-50% or about 50%, 30% or about 30%-45% or about 45%, 30% or about 30%-40% or about 40%, 30% or about 30%-35% or about 35%, 35% or about 35%-50% or about 50%, 35% or about 35%-45% or about 45%, 35% or about 35%-40% or about 40%, 40% or about 40%-50% or about 50%, 40% or about 40%-45% or about 45%, or 45% or about 45%-50% or about 50% w/w dextran, e.g., dextran 40. In some embodiments, the dextran solution comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% w/w dextran, e.g., dextran 40. In some embodiments, the dextran solution comprises about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% w/w dextran, e.g., dextran 40.

In some embodiments, the dextran solution contains dextran, e.g., dextran 40, at or about 25 g/l to 200 or about 200 g/l. In some embodiments, the dextran solution comprises dextran, e.g., dextran 40, 35 or about 35-200 or about 200, 25 or about 25-175 or about 175, 25 or about 25-150 or about 150, 25 or about 25-125 or about 125, 25 or about 25-100 or about 100, 25 or about 25-75 or about 75, 25 or about 25-50 or about 50, 50 or about 50-200 or about 200, 50 or about 50-175 or about 175, 50 or about 50-150 or about 150, 50 or about 50-125 or about 125, 50 or about 50-100 or about 100, 50 or about 50-75 or about 75, 75 or about 75-200 or about 200, 75 or about 75-175 or about 175, 75 or about 75-150 or about 150, 75 or about 75-125 or about 125, 75 or about 75-100 or about 100, 100 or about 100-200 or about 200, 100 or about 100-175 or about 175, 100 or about 100-150 or about 150, 100 or In some embodiments, the dextran solution comprises about 100-125 or about 125, 125 or about 125-200 or about 200, 125 or about 125-175 or about 175, 125 or about 125-150 or about 150, 150 or about 150-200 or about 200, 150 or about 150-175 or about 175, or 175 or about 175-200 or about 200 g/l. In some embodiments, the dextran solution comprises 25, 50, 75, 100, 125, 150, 175 or 200 g/l of dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises 100 g/l of dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 25, about 50, about 75, about 100, about 125, about 150, about 175, or about 200 g/l of dextran, e.g., Dextran 40. In some embodiments, the dextran solution comprises about 100 g/l of dextran, e.g., Dextran 40.

In some embodiments, the dextran solution additionally comprises glucose (also referred to as dextrose). In some embodiments, the dextran solution comprises 10 or about 10 g/l to 100 or about 100 g/l of glucose. In some embodiments, the dextran solution comprises 10 or about 10-100 or about 100, 10 or about 10-90 or about 90, 10 or about 10-80 or about 80, 10 or about 10-70 or about 70, 10 or about 10-60 or about 60, 10 or about 10-50 or about 50, 10 or about 10-40 or about 40, 10 or about 10-30 or about 30, 10 or about 10-20 or about 20, 20 or about 20-100 or about 100, 20 or about 20 ~90 or about 90, 20 or about 20-80 or about 80, 20 or about 20-70 or about 70, 20 or about 20-60 or about 60, 20 or about 20-50 or about 50, 20 or about 20-40 or about 40, 20 or about 20-30 or about 30, 30 or about 30-100 or about 100, 30 or about 30-90 or about 90, 30 or about 30-80 or about 80, 30 or about 30-70 or about 70, 30 or about 30-60 or about 60, 30 or about 30-50 or about 50, 30 or about 30-40 or about 40, 40 or about 40-100 or about 100, 40 or about 40-90 or about 90, 40 or about 40-80 or about 80, 40 or about 40-70 or about 70, 40 or about 40-60 or about 60, 40 or about 40-50 or about 50, 50 or about 50-100 or about 100, 50 or about 50-90 or about 90, 50 or about 50-80 or about 80, 50 or about 50-70 or about 70, 50 or about 50-60 or about 60, 60 or about 60-100 or about 100, 60 or about 60-90 or about 90, 60 or about 60-80 or about 80, 60 or about 60-70 or about 70, 70 or about 70-100 or about 100, 70 or about 70-90 or about 90, 70 or about 70-80 or about 80, 80 or about 80-90 or about 90, 80 or about 80-100 or about 100, 80 or about 80-90 or about 90, or 90 or about 90-100 or about 100 g/l of glucose. In some embodiments, the dextran solution comprises 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 g/l of glucose. In some embodiments, the dextran solution comprises 50 g/l of glucose. In some embodiments, the dextran solution comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 g/l of glucose. In some embodiments, the dextran solution comprises 50 g/l of glucose.

In some embodiments, the dextran solution is composed of dextran, e.g., dextran 40, and glucose in water.

In some embodiments, the cryopreservation composition comprises 10% or about 10% v/v to 50% or about 50% v/v of a dextran solution described herein. In some embodiments, the cryopreservation composition comprises 10% or about 10% to 50% of a dextran solution, e.g., a dextran solution described herein, 10% or about 10% to 45% or about 45%, 10% or about 10% to 40% or about 40%, 10% or about 10% to 35% or about 35%, 10% or about 10% to 30% or about 30%, 10% or about 10% to 25% or about 25%, 10% or about 10% to 20% or about 20%, 10% or about 10% to 15% or about 15% of a dextran solution, e.g., a dextran solution described herein. or about 15%, 15% or about 15% to 50% or about 50%, 15% or about 15% to 45% or about 45%, 15% or about 15% to 40% or about 40%, 15% or about 15% to 35% or about 35%, 15% or about 15% to 30% or about 30%, 15% or about 15% to 25% or about 25%, 15% or about 15% to 20% or about 20%, 20% or about 20% to 50% or about 50%, 20% or about 20% to 45% or about 45%, 20% or is about 20% to 40% or about 40%, 20% or about 20% to 35% or about 35%, 20% or about 20% to 30% or about 30%, 20% or about 20% to 25% or about 25%, 25% or about 25% to 50% or about 50%, 25% or about 25% to 45% or about 45%, 25% or about 25% to 40% or about 40%, 25% or about 25% to 35% or about 35%, 25% or about 25% to 30% or about 30%, 30% or about 30% to 50% or In some embodiments, the cryopreservation composition comprises a dextran solution, e.g., a dextran solution described herein, at 10%, 15%, 20%, 25%, 30%, 35%, 40%, or about 50% v/v. In some embodiments, the cryopreservation composition comprises a dextran solution, e.g., a dextran solution described herein, at 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% v/v. In some embodiments, the cryopreservation composition comprises a dextran solution, e.g., a dextran solution described herein, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% v/v.

In some embodiments, the cryopreservation composition comprises 10 or about 10-50 or about 50 g/l of dextran, e.g., dextran 40. In some embodiments, the cryopreservation composition comprises 10 or about 10-50 or about 50, 10 or about 10-45 or about 45, 10 or about 10-40 or about 40, 10 or about 10-35 or about 35, 10 or about 10-30 or about 30, 10 or about 10-25 or about 25, 10 or about 10-20 or about 20, 10 or about 10-15 or about 15, 15 or about 15-50 or about 50, 15 or about 15-45 or about 45, 15 or about 15-40 or about 40, 15 or about 15-35 or about 35, 15 or about 15-30 or about 30, 15 or about 15-25 or about 25, 15 or about 15-20 or about 20, 20 or about 20-50 or about 50, 20 or about 20-4 5 or about 45, 20 or about 20-40 or about 40, 20 or about 20-30 or about 30, 20 or about 20-25 or about 25, 25 or about 25-50 or about 50, 25 or about 25-45 or about 45, 25 or about 25-40 or about 40, 25 or about 25-35 or about 35, 25 or about 25-30 or about 30, 30 or about 30-50 or about 50, 30 or about 30-45 or about 45, 30 or about 30-40 or about 40, 30 or about 30-35 or about 35, 35 or about 35-50 or about 50, 35 or about 35-45 or about 45, 35 or about 35-40 or about 40, 40 or about 40-50 or about 50, 40 or about 40-45 or about 45, or 45 or about 45-50 or about 50 g/l. In some embodiments, the cryopreservation composition comprises 10, 15, 20, 25, 30, 30, 35, 40, 45 or 50 g/l of dextran, e.g., dextran 40. In some embodiments, the cryopreservation composition comprises about 10, about 15, about 20, about 25, about 30, about 30, about 35, about 40, about 45, or about 50 g/l of dextran, e.g., dextran 40.

3. Glucose In some embodiments, the cryopreservation composition comprises glucose.

In some embodiments, as described herein, the cryopreservation composition comprises a dextran solution containing glucose.

In some embodiments, the cryopreservation composition includes a dextran solution that does not include glucose. In some embodiments, for example, when the dextran solution does not include glucose, glucose is added separately to the cryopreservation composition.

In some embodiments, the cryopreservation composition comprises 5 or about 5-25 or about 25 g/l of glucose. In some embodiments, the cryopreservation composition comprises 5 or about 5-25 or about 25, 5 or about 5-20 or about 20, 5 or about 5-15 or about 15, 5 or about 5-10 or about 10, 10 or about 10-25 or about 25, 10 or about 10-20 or about 20, 10 or about 10-15 or about 15, 15 or about 15-25 or about 25, 15 or about 15-20 or about 20, or 20 or about 20-25 or about 25 g/l of glucose. In some embodiments, the cryopreservation composition comprises 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 or 25 g/l of glucose. In some embodiments, the cryopreservation composition comprises 12.5 g/l of glucose. In some embodiments, the cryopreservation composition comprises about 5, about 7.5, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, or about 25 g/l of glucose. In some embodiments, the cryopreservation composition comprises about 12.5 g/l of glucose.

In some embodiments, the cryopreservation composition comprises glucose at less than 2.75% w/v. In some embodiments, the cryopreservation composition comprises glucose at less than 27.5 g/l. In some embodiments, the cryopreservation composition comprises glucose at less than 2% w/v. In some embodiments, the cryopreservation composition comprises glucose at less than 1.5% w/v. In some embodiments, the cryopreservation composition comprises about 1.25% w/v or less.

4. Dimethyl Sulfoxide In some embodiments, the cryopreservation composition comprises dimethyl sulfoxide (DMSO, also known as methyl sulfoxide and methylsulfinylmethane).

In some embodiments, the DMSO is provided as a solution, also referred to herein as a DMSO solution. Thus, in some embodiments, the cryopreservation composition comprises a DMSO solution.

In some embodiments, the DMSO solution is suitable for intravenous use.

In some embodiments, the DMSO solution comprises 1.1 g/ml DMSO. In some embodiments, the DMSO solution comprises about 1.1 g/ml DMSO.

In some embodiments, the cryopreservation composition comprises 1% or about 1% to 10% or about 10% v/v of a DMSO solution. In some embodiments, the cryopreservation composition comprises 1% or about 1% to 10% or about 10%, 1% or about 1% to 9% or about 9%, 1% or about 1% to 8% or about 8%, 1% or about 1% to 7% or about 7%, 1% or about 1% to 6% or about 6%, 1% or about 1% to 5% or about 5%, 1% or about 1% to 4% or about 4%, 1% or about 1% to 3% or about 3%, 1% or about 1% to 2% or about 2%, 2% or about 2% to 10% or about 10 ... % or about 2% to 9% or about 9%, 8% or about 8%, 2% or about 2% to 7% or about 7%, 2% or about 2% to 6% or about 6%, 2% or about 2% to 5% or about 5%, 2% or about 2% to 4% or about 4%, 2% or about 2% to 3% or about 3%, 3% or about 3% to 10% or about 10%, 3% or about 3% to 9% or about 9%, 3% or about 3% to 8% or about 8%, 3% or about 3% to 7% or about 7%, 3% or about 3% to 6% or about 6%, 3% or about 3% to 5% or about 5%, 3% or about 3% to 4% or about 4%, 4% or about 4% to 10% or about 10%, 4% or about 4% to 9% or about 9%, 4% or about 4% to 8% or about 8%, 4% or about 4% to 7% or about 7%, 4% or about 4% to 6% or about 6%, 4% or about 4% to 5% or about 5%, 5% or about 5% to 10% or about 10%, 5% or about 5% to 9% or about 9%, 5% or about 5% to 8% or about 8%, 5% or about 5% to 7% or about 7%, 5% or about 5% to 6% or about 6%, 6% or about 6% to 10% or about 10%, 6% or about 6% to 9% or about 9%, 6% or about 6% to 8% or about 8%, 6% or about 6% to 7% or about 7%, 7% or about 7% to 10% or about 10%, 7% or about 7% to 9% or about 9%, 7% or about 7% to 8% or about 8%, 8% or about 8% to 10% or about 10%, 8% or about 8% to 9% or about 9%, or 9% or about 9% to 10% or about 10% v/v. In some embodiments, the cryopreservation composition comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% v/v DMSO solution. In some embodiments, the cryopreservation composition comprises 5% DMSO solution. In some embodiments, the cryopreservation composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% v/v DMSO solution. In some embodiments, the cryopreservation composition comprises about 5% DMSO solution.

In some embodiments, the cryopreservation composition comprises 11 or about 11-110 or about 110 g/l of DMSO. In some embodiments, the cryopreservation composition comprises 11 or about 11-110 or about 110, 11 or about 11-99 or about 99, 11 or about 11-88 or about 88, 11 or about 11-77 or about 77, 11 or about 11-66 or about 66, 11 or about 11-55 or about 55, 11 or about 11-44 or about 44, 11 or about 11-33 or about 33, 11 or about 11-22 or about 22, 22 or about 22-110 or about 110, 22 or about 22-99 or about 99, 22 or about 22-88 or about 88, 22 or about 22-77 or about 77, 22 or about 22-77 or about 77, 22 or about 22-66 or about 66, 22 or about 22-55 or about 55, 22 or about 22-44 or about 44, 22 or about 22-33 or about 33, 33 or about 33-110 or about 110, 33 or about 33-99 or about 99, 33 or about 33-88 or about 88, 33 or about 33-77 or about 77, or about 33 to 66 or about 66, 33 or about 33 to 55 or about 55, 33 or about 33 to 44 or about 44, 44 or about 44 to 110 or about 110, 44 or about 44 to 99 or about 99, 44 or about 44 to 88 or about 88, 44 or about 44 to 77 or about 77, 44 or about 44 to 66 or about 66, 44 or about 44 to 55 or about 55, 55 or about 55 to 110 or about 110, 55 or about 55 to 99 or about 99, 55 or about 55 to 88 or about 8 In some embodiments, the cryopreservation composition comprises 11, 22, 33, 44, 55, 66, 77, 88, 99, or 110 g/l of DMSO. In some embodiments, the cryopreservation composition comprises 55 g/l of DMSO. In some embodiments, the cryopreservation composition comprises about 11, about 22, about 33, about 44, about 55, about 66, about 77, about 88, about 99, or about 110 g/l of DMSO. In some embodiments, the cryopreservation composition comprises about 55 g/l of DMSO.

5. Buffer In some embodiments, the cryopreservation composition comprises a buffer solution, for example a buffer solution suitable for intravenous administration.

Buffer solutions include, but are not limited to, phosphate buffered saline (PBS), Ringer's solution, Tyrode's buffer, Hank's balanced salt solution, Earle's balanced salt solution, saline, and Tris.

In some embodiments, the buffer solution is phosphate buffered saline (PBS).

6. Exemplary Cryopreservation Compositions In some embodiments, the cryopreservation composition comprises or is comprised of the following components: 1) albumin, e.g., human albumin, 2) dextran, e.g., dextran 40, 3) DMSO, and 4) a buffer solution. In some embodiments, the cryopreservation composition additionally comprises glucose. In some embodiments, the cryopreservation composition is comprised of 1) albumin, e.g., human albumin, 2) dextran, e.g., dextran 40, 3) glucose, 4) DMSO, and 5) a buffer solution.

In some embodiments, the cryopreservation composition comprises 1) an albumin solution as described herein, 2) a dextran solution as described herein, 3) a DMSO solution as described herein, and 4) a buffer solution.

In some embodiments, the cryopreservation composition comprises 1) an albumin solution as described herein, 2) a dextran solution as described herein, 3) a DMSO solution as described herein, and 4) a buffer solution.

In some embodiments, the cryopreservation composition does not include cell culture medium.

In one embodiment, the cryopreservation composition comprises about 40 mg/ml human albumin, 25 mg/ml dextran 40, 12.5 mg/ml glucose and 55 mg/ml DMSO.

In one embodiment, the cryopreservation composition comprises or consists of approximately 40 mg/ml human albumin, 25 mg/ml dextran 40, 12.5 mg/ml glucose, 55 mg/ml DMSO, and 0.5 mg/ml 100% phosphate buffered saline (PBS) in water.

In one embodiment, the cryopreservation composition comprises about 32 mg/ml human albumin, 25 mg/ml dextran 40, 12.5 mg/ml glucose and 55 mg/ml DMSO.

In one embodiment, the cryopreservation composition comprises or consists of approximately 32 mg/ml human albumin, 25 mg/ml dextran 40, 12.5 mg/ml glucose, 55 mg/ml DMSO, and 0.54 mg/ml 100% phosphate buffered saline (PBS) in water.

Examples of cryopreservation compositions are shown in Table 3.

B. Cryopreservation Methods The cryopreservation compositions described herein can be used to cryopreserve cells, e.g., therapeutic cells, e.g., natural killer (NK) cells, e.g., the NK cells described herein.

In some embodiments, the cell is an animal cell. In some embodiments, the cell is a human cell.

In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is selected from a basophil, an eosinophil, a neutrophil, a mast cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a natural killer cell, a B cell, a T cell, and combinations thereof.

In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the natural killer cells are expanded and stimulated by the methods described herein.

In some embodiments, cryopreservation of cells includes mixing the cells with a cryopreservation composition described herein or a component thereof to produce a composition, e.g., a pharmaceutical composition; and freezing the mixture.

In some embodiments, cryopreservation of cells includes mixing a composition comprising cells with a cryopreservation composition or a component thereof described herein to produce a composition, e.g., a pharmaceutical composition; and freezing the mixture. In some embodiments, the composition comprising cells includes cells and a buffer. Suitable buffers are described herein.

In some embodiments, cryopreservation of cells includes mixing a composition comprising cells and a buffer, e.g., PBS, with a composition comprising albumin, dextran, and DMSO, e.g., as described herein; and freezing the mixture.

In some embodiments, cryopreservation of cells includes mixing a composition comprising cells and a buffer, e.g., PBS, 1:1 with a composition comprising 40 mg/ml albumin, e.g., human albumin, 25 mg/ml dextran, e.g., dextran 40, 12.5 mg/ml glucose, and 55 mg/ml DMSO.

In some embodiments, a composition comprising cells and a buffer, e.g., PBS, comprises cells at or about 2× ^{10 7} to 2×10 ⁹ or about 2×10 ⁹ cells/ml. In some embodiments, a composition comprising cells and a buffer, e.g., PBS, comprises 2×10 ⁸ cells/ml. In some embodiments, a composition comprising cells and a buffer, e.g., PBS, comprises about 2×10 ⁸ cells/ml.

In some embodiments, cell cryopreservation includes mixing cells, a buffer, e.g., PBS, albumin, e.g., human albumin, dextran, e.g., dextran 40, and DMSO; freezing the mixture.

In some embodiments, the mixture comprises 1× ¹⁰⁷ or about 1× ¹⁰⁷ to 1× ¹⁰⁹ or about 1× ¹⁰⁹ cells/ml. In some embodiments, the mixture comprises 1× ¹⁰⁸ cells/ml. In some embodiments, the mixture comprises about 1× ¹⁰⁸ cells/ml.

Suitable albumin, dextran and DMSO ranges are described above.

In some embodiments, the composition is frozen at or below -135°C.

In some embodiments, the composition freezes at a controlled rate.

III. ANTIBODIES The methods described herein include administration of a CD20-targeting antibody. In some embodiments, the CD20-targeting antibody is a CD20-targeting antibody selected from Table 6, or a combination thereof.

In some embodiments, the CD20-targeting antibody is selected from obinutuzumab (or a biosimilar thereof), ofatumumab (or a biosimilar thereof), and combinations thereof.

IV. Pharmaceutical Compositions The present invention provides pharmaceutical compositions comprising the natural killer cells described herein, and dosage units of the pharmaceutical compositions described herein.

In some cases, the dosage unit contains between 100 million and 1.5 billion cells, e.g., 100 million, 200 million, 300 million, 400 million, 500 million, 600 million, 700 million, 800 million, 900 million, 1 billion, 1.1 billion, 1.2 billion, 1.3 billion, 1.4 billion, or 1.5 billion cells.

Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the term "pharma-ceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., suitable for pharmaceutical administration.

In some embodiments, the pharmaceutical composition comprises: a) a natural killer cell described herein; and b) a cryopreserved composition.

Suitable cryopreservation compositions are described herein.

In some embodiments, the composition is frozen. In some embodiments, the composition is frozen for at least 3 months, e.g., at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 24 months, or at least 36 months.

In some embodiments, at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% of the natural killer cells survive post-thawing.

In some embodiments, the pharmaceutical composition comprises: a) a cryopreserved composition described herein; and b) therapeutic cells.

In some embodiments, the therapeutic cell is an animal cell. In some embodiments, the therapeutic cell is a human cell.

In some embodiments, the therapeutic cell is an immune cell. In some embodiments, the immune cell is selected from a basophil, an eosinophil, a neutrophil, a mast cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a natural killer cell, a B cell, a T cell, and combinations thereof.

In some embodiments, the immune cells are natural killer (NK) cells. In some embodiments, the natural killer cells are expanded and stimulated by the methods described herein.

In some embodiments, the pharmaceutical composition additionally comprises c) a buffer solution. Suitable buffer solutions are, for example, as described herein for the cryopreserved composition.

In some embodiments, the pharmaceutical composition comprises cells at or about 1× ^{10 7} to 1×10 ⁹ cells/ml or about 1×10 ⁹ cells/ml. In some embodiments, the pharmaceutical composition comprises cells at 1×10 ⁸ cells/ml. In some embodiments, the pharmaceutical composition comprises cells at about 1×10 ⁸ cells/ml.

In some embodiments, the pharmaceutical composition additionally comprises an antibody or antigen-binding fragment thereof, e.g., an antibody described herein.

Pharmaceutical compositions are typically formulated for the intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

Methods for formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal or subcutaneous applications can contain components such as: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and tonicity regulators, such as sodium chloride or dextrose. The pH can be titrated with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can encompass sterile acids and sterile aqueous solutions (if water soluble) or dispersions for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid enough for easy injection. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to add isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, to the composition. Absorption of the injectable composition can be delayed by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization as required. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients from those enumerated above. In the case of sterile acid solutions for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

V. Methods of Treatment The NK cells described herein have utility in treating cancer or other proliferative disorders.

To this end, the present invention also provides a method of treating a patient suffering from a disorder, e.g., a cancer-related disorder, e.g., a CD20+ cancer, comprising administering an NK cell, e.g., an NK cell described herein, and a CD20-targeted antibody, e.g., an antibody described herein.

The present invention also provides a method for preventing, reducing and/or inhibiting recurrence, growth, proliferation, migration and/or metastasis of a cancer cell or population of cancer cells in an individual in need thereof, comprising administering NK cells, e.g., NK cells described herein, and a CD20-targeted antibody, e.g., an antibody described herein.

Furthermore, the present invention provides a method of enhancing, improving and/or increasing a response to an anti-cancer therapy in an individual in need thereof, comprising administering NK cells, e.g., NK cells described herein, and a CD20-targeted antibody, e.g., an antibody described herein.

The present invention also provides a method of inducing an immune system in an individual in need thereof, comprising administering NK cells, e.g., NK cells described herein, and a CD20-targeted antibody, e.g., an antibody described herein.

The methods described herein include methods of treating disorders associated with abnormal apoptotic or differentiation processes, e.g., cell proliferation or differentiation disorders, e.g., cancers such as solid tumors and hematopoietic cancers. Generally, the methods include administering a therapeutically effective amount of a therapeutic agent as described herein to an individual in need or determined to be in need of treatment. In some embodiments, the methods include administering a therapeutic agent comprising NK cells, e.g., NK cells described herein, and an XX-targeted antibody, e.g., an antibody described herein, in a therapeutically effective amount.

As used herein, the terms "treatment", "treat" and "treating" refer to the regression, alleviation, delayed onset or inhibition of progression of a disorder associated with abnormal apoptotic or differentiation processes. For example, treatment can achieve a reduction in tumor size or growth rate. The administration of a compound described herein in a therapeutically effective amount to treat a condition associated with abnormal apoptotic or differentiation processes may achieve, among other things, a reduction in tumor size or growth rate, a reduction in the risk or frequency of recurrence, a delay in recurrence, a reduction in metastasis, an increase in survival and/or a reduction in morbidity and mortality. In some embodiments, the therapeutic agent can be administered after one or more symptoms have developed. In other embodiments, the therapeutic agent may be administered in the absence of symptoms. For example, the therapeutic agent may be administered to a susceptible individual before symptoms have begun (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). The therapeutic agent can also continue to be administered after symptoms have resolved, e.g., to prevent or delay recurrence.

As used herein, the term "inhibit" refers to the inhibition of growth, division, maturation or viability of cancer cells and/or the induction of death of cancer cells, either individually or collectively with other cancer cells, in relation to cancer and/or cancer cell proliferation, by cytotoxicity, nutrient depletion or induction of apoptosis.

As used herein, "delaying" the progression of a disease or disorder, or one or more symptoms thereof, means to delay, prevent, slow, retard, stabilize, and/or postpone the onset of the disease, disorder, or symptoms thereof. The delay may be of varying lengths of time depending on the individual's history of the disease and/or treatment. As will be appreciated by those of skill in the art, a sufficient or significant delay encompasses prevention, in that the disease, disorder, or symptoms thereof do not, in effect, occur in the individual. For example, a method of "delaying" the onset of cancer is one that reduces the probability of the onset of the disease for a given time range and/or reduces the extent of the disease for a given time range, as compared to not utilizing the method. Such comparisons may be based on clinical trials using a statistically significant number of individuals.

As used herein, "prevention" or "preventing" refers to preventing a disease or disorder from initiating so that clinical symptoms of the disease do not occur. Thus, "prevention" refers to administering a therapy (e.g., administration of a therapeutic agent) to an individual before symptoms of the disease are detectable in the individual and/or before a particular stage of the disease is reached (e.g., administration of a therapeutic agent to a patient whose cancer has not yet metastasized). The individual may be at risk of developing a disease or disorder or at risk of disease progression, e.g., at risk of cancer metastasis, e.g., an individual who has one or more risk factors known to be associated with the onset or initiation of a disease or disorder. For example, the individual may carry a mutation associated with the onset or progression of cancer. Furthermore, it is understood that prevention does not achieve a complete prevention against the onset of a disease or disorder. In some cases, prevention includes a reduction in the risk of developing a disease or disorder. A reduction in risk may not achieve a complete elimination of the risk of developing a disease or disorder.

The degree of "increased" or "enhanced" (e.g., in relation to anti-tumor response, metastasis of cancer cells) refers to an increase of 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or higher (e.g., 100-fold, 500-fold, 1000-fold) relative to the amount or level referenced herein (including all integers and decimal values greater than 1 between the referenced integers, e.g., 2.1, 2.2, 2.3, 2.4, etc.). This can also encompass an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500% or at least 1000% relative to the amounts or levels mentioned herein.

A "reduced" or "lower" amount (e.g., in relation to tumor size, cancer cell proliferation or growth) refers to a reduction of 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or higher (e.g., 100-fold, 500-fold, 1000-fold) relative to the amount or level referenced herein (including all integers and decimal values greater than 1 between the referenced integers, e.g., 1.5, 1.6, 1.7, 1.8, etc.). This can also encompass a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 500% or at least 1000% relative to the amounts or levels mentioned herein.

A. Disorders The disclosed method and manufactured composition have applications for targeting a number of disorders, such as cell proliferation disorders.In the present invention, the advantage of such an approach is that allogeneic cells are used in combination with the administration of exogenous antibodies to target the specific proliferative cells targeted by the exogenous antibodies.By using such an approach and pharmaceutical composition of the present invention, it is possible to specifically target cells that exhibit potentially harmful proliferative activity without administering systemic drugs or toxins that indiscriminately affect proliferative cells, unlike existing therapies such as chemotherapy or radiation therapy.

Examples of cell proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders, e.g., leukemia. Metastatic tumors can originate from a variety of primary tumor types, including, but not limited to, prostate, colon, lung, breast, and hepatic origin.

As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or pathology characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states can be classified as pathological, i.e., characterized or diseased by a disease state, or non-pathological, i.e., not a normal state but not associated with a disease state. The term is meant to encompass all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples for non-pathological hyperproliferative cells include cell proliferation associated with wound healing.

The term "cancer" or "neoplasm" encompasses malignancies of various organ systems, such as those occurring in the lung, breast, thyroid, lymphatic system, gastrointestinal and genitourinary tract, as well as malignancies such as adenocarcinomas, e.g., most colon cancers, renal cell carcinoma, prostate and/or testicular tumors, non-small cell lung carcinoma, small intestine cancer and esophageal cancer.

The term "carcinoma" is understood in the art and refers to malignant tumors of epithelial or endocrine tissues, such as respiratory system carcinoma, gastrointestinal system carcinoma, genitourinary system carcinoma, testicular carcinoma, breast carcinoma, prostate carcinoma, endocrine system carcinoma, and melanoma. In some embodiments, the disease is renal cancer or melanoma. Examples of carcinomas include carcinomas arising from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also encompasses carcinosarcomatosis, including, for example, malignant tumors composed of carcinoma and sarcoma tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells originate from recognizable glandular structures.

The term "sarcoma" is understood in the art to refer to malignant tumors of mesenchymal origin.

Additional examples for proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorder" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., originating from the myeloid, lymphoid, or erythroid lineages or precursor cells thereof. Preferably, the disease arises from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Examples of additional myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to, ALL, including B-lineage acute lymphoblastic leukemia (ALL) and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphoblastic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Sternberg disease.

In some embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Kaposi's sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, typical teratoma/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid, cardiac tumor, medulloblastoma, germ cell tumor, primary CNS lymphoma, cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma in situ, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma , extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer (e.g., intraocular melanoma or retinoblastoma), fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, cardiac tumors, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, Pancreatic neuroendocrine tumors, renal (renal cell) carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung carcinoma, pleuropulmonary blastoma, and tracheobronchial tumors), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic carcinoma, metastatic squamous cell carcinoma, midline carcinoma tract carcinoma), oral cavity cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, myeloproliferative neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor, papillomatosis, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm, multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer cancer), primary malignant lymphoma of the central nervous system, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (e.g., childhood rhabdomyosarcoma, childhood vascular tumor, Ewing's sarcoma, Kaposi's sarcoma, osteosarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, tracheobronchial tumor, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and Wilms' tumor.

In some embodiments, the cancer is a solid tumor.

In some embodiments, the cancer is metastatic.

In some embodiments, the disorder is selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, follicular lymphoma, granulomatosis with polyangiitis, microscopic polyangiitis, multiple sclerosis, non-Hodgkin's lymphoma, pemphigus vulgaris, rheumatoid arthritis, and combinations thereof.

In some embodiments, the cancer is a CD20+ cancer.

In some embodiments, the CD20+ cancer is selected from the group consisting of non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL).

In some embodiments, the CD20+ cancer is selected from the group consisting of indolent non-Hodgkin's lymphoma or aggressive non-Hodgkin's lymphoma (NHL). In some embodiments, the CD20+ cancer is relapsed or refractory indolent NHL or aggressive NHL of B-cell origin. Aggressive and indolent subtypes include those listed in Table 7.

B. Patients Suitable patients for the compositions and methods of the present invention include those suffering from, diagnosed with, or suspected of suffering from a cell proliferative and/or differentiative disorder, e.g., cancer. Patients who have undergone the procedures referred to in the present invention generally respond better to the methods and compositions of the present invention, in part because the pharmaceutical compositions are allogeneic and generally identify the target cells with antibodies rather than targeting proliferating cells. Therefore, there are fewer off-target effects and patients are more likely to complete a therapeutic regimen without substantial adverse off-target effects.

In some embodiments, the therapeutic methods provided herein can be used to treat an individual (e.g., a human, monkey, dog, cat, mouse) diagnosed with or suspected of suffering from a cell proliferative and/or differentiative disorder, e.g., cancer. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human.

As used herein, an individual refers to a mammal, including, for example, a human.

In some embodiments, the mammal is selected from the group consisting of armadillo, donkey, bat, bear, beaver, cat, chimpanzee, cow, coyote, deer, dog, dolphin, elephant, fox, panda, gibbon, giraffe, goat, gopher, hedgehog, hippopotamus, horse, humpback whale, jaguar, kangaroo, koala, leopard, deer, llama, lynx, mole, monkey, mouse, narwhal, orangutan, stag beetle, otter, bull, pig, polar bear, porcupine, puma, rabbit, raccoon, rat, rhino, sheep, squirrel, tiger, walrus, weasel, wolf, zebra, goat, horse, and combinations thereof.

In some embodiments, the mammal is a human.

The individual, e.g., a human individual, may be a child, e.g., at or about 0 years old to at or about 14 years old. The individual may be a youth, e.g., at or about 15 years old to at or about 24 years old. The individual may be an adult, e.g., at or about 25 years old to at or about 64 years old. The individual may be an elderly individual, e.g., 65 years old or older.

In some embodiments, the individual may be a human who has developed one or more symptoms associated with a cell proliferation and/or differentiation disorder, e.g., cancer, e.g., a tumor. Various stages of cancer can be treated using any of the methods of treatment provided herein. For example, cancer stages include, but are not limited to, early stage, advanced, locally advanced, in remission, refractory, recurrent after remission, and progressive. In some embodiments, the individual is in an early stage of cancer. In other embodiments, the individual is in an advanced stage of cancer. In various embodiments, the individual is suffering from stage 1, stage 2, stage 3, or stage 4 cancer. The methods of treatment described herein can promote tumor reduction or shrinkage, reduce or inhibit tumor growth or cancer cell proliferation, and/or induce, increase, or promote tumor cell killing. In some embodiments, the individual is in cancer remission. The methods of treatment described herein can prevent or delay cancer metastasis or recurrence.

In some embodiments, the individual is at risk for developing, or has genetic or other (e.g., risk factors for), a diagnosed or undiagnosed cell proliferative and/or differentiative disorder, e.g., cancer.

As used herein, an "at risk" individual is an individual who is at risk for developing the condition to be treated, e.g., a cell proliferation and/or differentiation disorder, e.g., cancer. Generally, an "at risk" individual may or may not have detectable disease, and may or may not have developed detectable disease prior to the treatment methods described herein. "At risk" means that the individual has one or more so-called risk factors, which are measurable parameters associated with the development of a disease or condition and are known in the art. For example, an at risk individual may have one or more risk factors that are measurable parameters associated with the development of cancer. An individual who has such one or more risk factors has a higher probability of developing cancer than an individual who does not have such risk factors. Generally, risk factors can include, for example, age, sex, race, diet, history of previous disease, presence of precursor disease, genetic (e.g., hereditary) considerations, and environmental exposures. In some embodiments, individuals at risk of developing cancer include, for example, individuals who have had the disease among their relatives, and individuals who have a risk determined by genetic or biochemical marker analysis.

In conjunction with this, the individual may be undergoing one or more standard therapies, such as chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof. Thus, one or more kinase inhibitors may be administered before, during, or after chemotherapy, radiation therapy, immunotherapy, surgery, or a combination thereof.

In certain embodiments, the individual may be a human who is (i) substantially refractory to one or more chemotherapeutic agents, or (ii) has relapsed after being treated with chemotherapy, or both (i) and (ii). In some embodiments, the individual is refractory to two or more, three or more, or four or more chemotherapeutic agents (including standard or experimental chemotherapy).

In some embodiments, the patient is diagnosed with or has been diagnosed with a disorder selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma, follicular lymphoma, granulomatosis with polyangiitis, microscopic polyangiitis, multiple sclerosis, non-Hodgkin's lymphoma, pemphigus vulgaris, rheumatoid arthritis, and combinations thereof.

In some embodiments, the patient is diagnosed with or has been diagnosed with a CD20+ cancer.

In some embodiments, the patient has been diagnosed with or has been diagnosed with CD20+ cancer by immunohistochemical staining of a cancer biopsy or surgical sample. In some embodiments, the patient has been diagnosed with or has been diagnosed with CD20+ cancer by chromogenic in situ hybridization. In some embodiments, the patient has been diagnosed with or has been diagnosed with CD20+ cancer by fluorescent in situ hybridization of a cancer biopsy or surgical sample.

In some embodiments, the patient has been diagnosed with or has been diagnosed with a CD20+ cancer by genetic analysis, e.g., identification of a CD20 mutant cancer, e.g., a somatic mutation, e.g., a somatic mutation in the CD20 (MS4A1) gene.

In some embodiments, the patient has a cancer comprising one or more of the sets of mutations set forth in Table 8, an insertion or deletion polymorphism in the CD20 gene, a copy number mutation in the CD20 gene, a methylation mutation in the CD20 gene, or a combination thereof.

In some embodiments, the patient has a chromosomal translocation associated with the cancer, e.g., a CD20+ cancer. In some embodiments, the patient has a fusion gene associated with the cancer, e.g., a CD20+ cancer.

In some embodiments, the patient is refractory or has relapsed after treatment with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone), e.g., 4 or more cycles of R-CHOP as second line chemotherapy, e.g., ICE (ifosfamide, carboplatin and etoposide) or DHAP (dexamethasone, high dose Ara-C cytarabine and platinol) alone or in combination with an approved therapeutic mAb (e.g., rituximab).

C. Lymphocyte Depletion In some embodiments, the patient is lymphocyte depleted prior to treatment.

Exemplary lymphodepleting chemotherapy regimens, along with associated useful biomarkers, are described in WO 2016/191756 and WO 2019/079564, which are incorporated herein by reference in their entireties. In certain embodiments, the lymphodepleting chemotherapy regimen comprises administering to the patient cyclophosphamide (200 mg/ ^m2 /day to 2000 mg/ ^m2 /day) and fludarabine (20 mg/ ^m2 /day to 900 mg/ ^m2 /day).

In some embodiments, lymphocyte depletion comprises administration of cyclophosphamide at or about 250-500 or about 500 mg/ ^m2 , e.g., administration of cyclophosphamide at or about 250-500 or about 500, 250, 400, 500, about 250, about 400 or about 500 mg/ ^m2 .

In some embodiments, lymphocyte depletion comprises administration of fludarabine at or about 20 mg/m ² /day to 40 or about 40 mg/m ² /day, for example, 30 or about 30 mg/m ² /day.

In some embodiments, lymphodepletion includes administration of both cyclophosphamide and fludarabine.

In some embodiments, patients are lymphodepleted with intravenous administration of cyclophosphamide (250 mg/m ² /day) and fludarabine (30 mg/m ² /day).

In some embodiments, patients are lymphodepleted with intravenous administration of cyclophosphamide (500 mg/m ² /day) and fludarabine (30 mg/m ² /day).

In some embodiments, lymphodepletion is performed up to 5 days prior to administering the first dose of NK cells. In some embodiments, lymphodepletion is performed up to 7 days prior to administering the first dose of NK cells.

In some embodiments, lymphodepletion is performed daily for three consecutive days (i.e., days -5 to -3) beginning 5 days prior to the first administration of NK cells.

In some embodiments, lymphodepletion occurs on days -5, -4, and -3.

D. Administration 1. NK Cells In some embodiments, NK cells are administered as part of a pharmaceutical composition, e.g., a pharmaceutical composition described herein. The cells are administered after thawing, optionally without any additional manipulation if the cryoprotectant is suitable for extemporaneous administration. For a given individual, a treatment regimen often involves administering multiple aliquots or doses of NK cells over time, often from a common batch or donor. In some embodiments, NK cells, e.g., NK cells described herein, are administered at or about 1×10 ⁸ to 8×10 9 or about 8×10 ⁹ NK cells per dose. In some embodiments, NK cells are administered at or about 1×10 ⁸ , 1×10 ⁹ or about 1×10 ⁹ , 4×10 ⁹ or about 4× ^{10 9 ,} or 8×10 ⁹ or about 8×10 ^{9 cells per} dose.

In some embodiments, the NK cells are administered weekly. In some embodiments, the NK cells are administered weekly or about weekly. In some embodiments, the NK cells are administered 8 weeks or about 8 weeks.

In some embodiments, the NK cells are cryopreserved in an infusion-ready media, e.g., a cryopreservation composition suitable for intravenous administration, as described herein.

In some embodiments, the NK cells are cryopreserved in vials containing 1×10 ⁸ or about 1×10 ⁸ to 8×10 ⁹ or about 8×10 ⁹ cells per vial. In some embodiments, the NK cells are cryopreserved in single dose vials.

In some embodiments, the cells are thawed prior to administration, e.g., in a 37°C water bath.

In some embodiments, the thawed vial of NK cells is aseptically transferred to a single dose container, e.g., a dose bag, e.g., using a vial adapter and a sterile syringe. The NK cells can be administered from the container to the patient as an IV infusion via a Y-type blood/solution set filter by gravity.

In some embodiments, the NK cells are administered as soon as possible after thawing, preferably within 90 minutes, e.g., within 80 minutes, within 70 minutes, within 60 minutes, within 50 minutes, within 40 minutes, within 30 minutes, within 20 minutes, or within 10 minutes. In some embodiments, the NK cells are administered within 30 minutes after thawing.

In some embodiments, the pharmaceutical composition is administered intravenously via a syringe.

In some embodiments, the patient is administered 1 ml, 4 ml, or 10 ml of the medication intravenously via a syringe.

2. Antibodies In some embodiments, the NK cells described herein, e.g., a pharmaceutical composition comprising the NK cells referred to herein, are administered in combination with an antibody, e.g., an antibody described herein, e.g., a CD20 antibody. In some embodiments, the antibody is administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the antibody is administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition. The antibody may be administered before, after, or simultaneously with the administration of the NK cells.

In some embodiments, the antibody is administered prior to administration of the NK cells. In some embodiments, the antibody is administered after administration of the NK cells.

In some embodiments, the NK cells are administered at least 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, or 240 minutes after completing antibody administration.

In some embodiments, the NK cells are administered the day after the antibody is administered.

In some embodiments, NK cells are administered at each dose and the antibody is administered only for a portion of the doses. For example, in some embodiments, NK cells are administered once per week and the antibody is administered once per month.

In some embodiments, the antibody is administered weekly for 8 weeks. In some embodiments, the antibody is administered every 2 weeks for 8 weeks.

In some embodiments, a dose of the antibody is provided prior to the first administration of the cells. In some embodiments, a reduced dose of the antibody is provided prior to the first administration of the cells.

3. Cytokines In some embodiments, a cytokine is administered to the patient.

In some embodiments, the cytokines are administered together with the NK cells as part of a pharmaceutical composition. In some embodiments, the cytokines are administered separately from the NK cells, e.g., as part of a separate pharmaceutical composition.

In some embodiments, the cytokine is IL-2.

In some embodiments, IL-2 is administered subcutaneously.

In some embodiments, IL-2 is administered 1-4 hours or about 1 to about 4 hours after administration of NK cells is completed. In some embodiments, IL-2 is administered at least 1 hour after administration of NK cells is completed. In some embodiments, IL-2 is administered within up to 4 hours after administration of NK cells is completed. In some embodiments, IL-2 is administered within a minimum of 1 hour and a maximum of 4 hours after administration of NK cells is completed.

In some embodiments, IL-2 is administered at up to 10,000,000 IU/m ² , for example up to 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000 or 10,000,000 IU/m ² .

In some embodiments, IL-2 is administered at or about 1,000,000, 2,000,000 or about 2,000,000, or 3,000,000 or about 3,000,000, or 4,000,000 or about 4,000,000, or 5,000,000 or about 5,000,000, or 6,000,000 or about 6,000,000, or 7,000,000 or about 7,000,000, or 8,000,000 or about 8,000,000, or 9,000,000 or about 9,000,000, or 10,000,000 IU/ ^m2 .

In some embodiments, IL-2 is administered at or about 1×10 ⁶ IU/m ^2. In some embodiments, IL- ^{2 is} administered at or about 2× ^{10 6 IU} /m ² .

In some embodiments, IL-2 is administered to the patient at less than 1×10 ⁶ IU/m ² .

In some embodiments, IL-2 is administered to the patient in a flat dose. In some embodiments, a flat dose of 6,000,000 IU or about 6,000,000 IU is administered to the patient.

In some embodiments, IL-2 is not administered to the patient.

E. An "effective amount" administered is an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic content is an amount that achieves a desired therapeutic effect. Such an amount may be the same as or different from the amount necessary to prevent the onset of a disease or disease symptoms, i.e., a prophylactically effective amount. An effective amount is administered in one or more administrations, applications or dosages. The therapeutically effective amount (i.e., an effective dosage) of a therapeutic compound is determined by the therapeutic compound selected. The composition may be administered one or more times per day or one or more times per week, including once at two-day intervals. Those skilled in the art will recognize that certain factors may affect the dosage and time required to effectively treat an individual, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the individual, and other diseases present. In conjunction with this, treating an individual with a therapeutically effective amount of a therapeutic compound according to the present invention may include a single treatment or a series of treatments.

The dosage, toxicity and therapeutic efficacy of therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50/ED50. Compounds with high therapeutic indices are preferred. Compounds with toxic side effects may be used, but care must be taken to design systems that target delivery of such compounds to diseased tissue sites in order to minimize potential damage to non-infected cells and reduce side effects.

Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds may be within a circulating concentration range that includes the ED50 with little or no toxicity. Dosages may vary within such ranges depending on the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective amount can be estimated initially in cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves half the maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

F. Combination Therapies In some embodiments, the methods include administering the NK cell and CD20 targeted antibodies described herein in combination with other therapies, e.g., additional antibodies, NK cell engagers, antibody drug conjugates (ADCs), chemotherapeutic drugs, e.g., small molecule drugs, immune checkpoint inhibitors, and combinations thereof.

1. Small Molecule/Chemotherapy Drugs In some embodiments, the additional therapy is a small molecule drug. In some embodiments, the additional therapy is a chemotherapy drug. In some embodiments, the additional therapy is a small molecule chemotherapy drug. Such small molecule drugs can include current standard regimens to which adaptive NK cell therapy is added. In some cases, the use of NK cells as described herein can enhance the effect of small molecule drugs, such as by enhancing the efficacy of the small molecule drug, reducing the amount of the small molecule drug required to achieve a desired effect, or reducing the toxicity of the small molecule drug.

In some embodiments, the drug is selected from the group consisting of:

In some embodiments, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4-acetyloxy-1,9,12-trihydroxy-15-[(2R,3S)-2-hydroxy-3-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoyl]oxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0 ^3,10 . ^{0 4,7} ]heptadeth-13-en-2-yl]benzoate (docetaxel) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is [(1S,2S,3R,4S,7R,9S,10S,12R,15S)-4,12-diacetyloxy-15-[(2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoyl]oxy-1,9-dihydroxy-10,14,17,17-tetramethyl-11-oxo-6-oxatetracyclo[11.3.1.0 ^3,10 . ^{0 4,7} ]heptadeth-13-en-2-yl]benzoate (paclitaxel) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is 6-N-(4,4-dimethyl-5H-1,3-oxazol-2-yl)-4-N-[3-methyl-4-([1,2,4]triazolo[1,5-a]pyridin-7-yloxy)phenyl]quinazoline-4,6-diamine (tucatinib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is an azanide; cyclobutane-1,1-dicarboxylic acid; platinum(2+) (carboplatin) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is methyl (1R,9R,10S,11R,12R,19R)-11-acetyloxy-12-ethyl-4-[(12S,14R)-16-ethyl-12-methoxycarbonyl-1,10-diazatetracyclo[12.3.1.0 ^3,11 . ^{0 4,9} ]octadeca-3(11),4,6,8,15-pentaen-12-yl]-10-hydroxy-5-methoxy-8-methyl-8,16-diazapentacyclo[10.6.1.0 ^1,9 . ^{0 2,7} . 0 ^16,19 ]nonadeca-2,4,6,13-tetraene-10-carboxylate (vinorelbine) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2-methylsulfonylethylamino)methyl]furan-2-yl]quinazolin-4-amine (lapatinib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is (E)-N-[4-[3-chloro-4-(pyridin-2-ylmethoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide (neratinib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is 6-acetyl-8-cyclopentyl-5-methyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrido[2,3-d]pyrimidin-7-one (palbociclib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is 7-cyclopentyl-N,N-dimethyl-2-[(5-piperazin-1-ylpyridin-2-yl)amino]pyrrolo[2,3-d]pyrimidine-6-carboxamide (ribociclib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is N-[5-[(4-ethylpiperazin-1-yl)methyl]pyridin-2-yl]-5-fluoro-4-(7-fluoro-2-methyl-3-propan-2-ylbenzimidazol-5-yl)pyrimidin-2-amine (abemaciclib) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-[(2R)-1-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]propan-2-yl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.0 ^4,9 ]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone (everolimus) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide (alpelisib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is 4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]methyl]-2H-phthalazin-1-one (olaparib) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (11S,12R)-7-fluoro-11-(4-fluorophenyl)-12-(2-methyl-1,2,4-triazol-3-yl)-2,3,10-triazatricyclo[7.3.1.0 ^5,13 ]trideca-1,5(13),6,8-tetraen-4-one (talazoparib) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is N-[2-[2-(dimethylamino)ethyl-methylamino]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide (osimertinib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (gefitinib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is (E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide (afatinib) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is azan; dichloroplatinum (cisplatin, platinol) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is an azanide; cyclobutane-1,1-dicarboxylic acid; platinum(2+) (carboplatin) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is 4-amino-1-[(2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one (gemcitabine) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is (2S)-2-[[4-[2-(2-amino-4-oxo-3,7-dihydropyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]amino]pentanedioic acid (pemetrexed) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is N,N-bis(2-chloroethyl)-2-oxo-1,3,2λ ⁵ -oxazaphosphinan-2-amine (cyclophosphamide) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (2R,3S,4S,5R)-2-(6-amino-2-fluoropurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol (fludarabine) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione (doxorubicin) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is methyl (1R,9R,10S,11R,12R,19R)-11-acetyloxy-12-ethyl-4-[(13S,15S,17S)-17-ethyl-17-hydroxy-13-methoxycarbonyl-1,11-diazatetracyclo[13.3.1.0 ^4,12 . 0 ^5,10 ]nonadeca-4(12),5,7,9-tetraen-13-yl]-8-formyl-10-hydroxy-5-methoxy-8,16-diazapentacyclo[10.6.1.0 ^1,9 . ^{0 2,7} . 0 ^16,19 ]nonadeca-2,4,6,13-tetraene-10-carboxylate (vincristine) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (8S,9S,10R,13S,14S,17R)-17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,12,14,15,16-octahydrocyclopenta[a]phenanthrene-3,11-dione (prednisone) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is N,3-bis(2-chloroethyl)-2-oxo-1,3,2λ ⁵ -oxazaphosphinan-2-amine (ifosfamide) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (5S,5aR,8aR,9R)-5-[[(2R,4aR,6R,7R,8R,8aS)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5H-[2]benzofuro[6,5-f][1,3]benzodioxol-8-one (etopside) or a pharma- ceutical acceptable salt thereof.

In some embodiments, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one (dexamethasone) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the drug is (8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one (cytarabine) or a pharma- ceutically acceptable salt thereof.

In some embodiments, the NK cells, e.g., the NK cells described herein, e.g., AB-101 cells, are administered in combination with a therapy selected from the group consisting of cyclophosphamide, doxorubicin, vincristine, prednisone, dexamethasone, cytarabine, e.g., high dose Ara-C cytarabine, platinol, and combinations thereof (e.g., R-CHOP, ICE, or DHAP), as well as a CD20-targeted antibody.

2. NK Cell Engagers In some embodiments, the additional therapy is an NK cell engager, for example a bispecific or trispecific antibody.

In some embodiments, the NK cell engager is a bispecific antibody against CD16 and a disease-associated antigen, e.g., a cancer-associated antigen, e.g., a cancer antigen described herein, e.g., CD20. In some embodiments, the NK cell engager is a trispecific antibody against CD16 and two disease-associated antigens, e.g., a cancer-associated antigen, e.g., an antigen described herein.

3. Checkpoint Inhibitors In some embodiments, the additional therapy is an immune checkpoint inhibitor.

In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and combinations thereof.

In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a VISTA inhibitor, a BTLA inhibitor, a TIM-3 inhibitor, a KIR inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD-96 inhibitor, a SIRPα inhibitor, and combinations thereof.

In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 (CD223) inhibitors, TIM-3 inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, A2aR inhibitors, CD73 inhibitors, NKG2A inhibitors, PVRIG/PVRL2 inhibitors, CEACAM1 inhibitors, CEACAM5 inhibitors, CEACAM6 inhibitors, FAK inhibitors, CCL2 inhibitors, CCR2 inhibitors, LIF inhibitors, CD47 inhibitors, SIRPα inhibitors, CSF-1 inhibitors, M-CSF inhibitors, CSF-1R inhibitors, IL-1 inhibitors, IL-1R3 inhibitors, IL-RAP inhibitors, IL-8 inhibitors, SEMA4D inhibitors, Ang-2 inhibitors, CELVER-1 inhibitors, Axl inhibitors, phosphatidylserine inhibitors, and combinations thereof.

In some embodiments, the immune checkpoint inhibitor is selected from the substances listed in Table 9, or combinations thereof.

In some embodiments, the immune checkpoint inhibitor is an antibody.

In some embodiments, the PD-1 is selected from the group consisting of pembrolizumab, nivolumab, toripalimab, cemiplimab-rwlc, sintilimab, and combinations thereof.

In some embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, durvalumab, avelumab, and combinations thereof.

In some embodiments, the CTLA-4 inhibitor is ipilimumab.

In some embodiments, the PD-1 inhibitor is selected from the group of inhibitors shown in Table 10.

In some embodiments, the PD-L1 inhibitor is selected from the group of inhibitors shown in Table 11.

In some embodiments, the CTLA-4 inhibitor is selected from the group of inhibitors shown in Table 12.

In some embodiments, the immune checkpoint inhibitor is a small molecule drug. Small molecule checkpoint inhibitors are described, for example, in WO2015/034820A1, WO2015/160641A2, WO2018/009505A1, WO2017/066227A1, WO2018/044963A1, WO2018/026971A1, WO2018/045142A1, WO2018/005374A1, WO2017/202275A1, WO2017/202273A1, WO2017/202276A1, W WO2018/006795A1, WO2016/142852A1, WO2016/142894A1, WO2015/033301A1, WO2015/033299A1, WO2016/142886A2, WO2016/142833A1, WO2018/051255A1, WO2018/051254A1, WO2017/205464A1, US2017/0107216A1, WO2017/070089A1, WO2017 /106634A1, US2017/0174679A1, US2018/0057486A1, WO2018/013789A1, US2017/0362253A1, WO2017/192961A1, WO2017/118762A1, US2014/199334A1, WO2015/036927A1, US2014/0294898A1, US2016/0340391A1, WO2016/039749A1, WO2017 /176608A1, WO2016/077518A1, WO2016/100608A1, US2017/0252432A1, WO2016/126646A1, WO2015/044900A1, US2015/0125491A1, WO2015/033303A1, WO2016/142835A1, WO2019/008154A1, WO2019/008152A1 and WO2019023575A1.

In some embodiments, the PD-1 inhibitor is 2-[[4-amino-1-[5-(1-amino-2-hydroxypropyl)-1,3,4-oxadiazol-2-yl]-4-oxobutyl]carbamoylamino]-3-hydroxypropanoic acid (CA-170).

In some embodiments, the immune checkpoint inhibitor is (S)-1-(3-bromo-4-((2-bromo-[1,1'-biphenyl]-3-yl)methoxy)benzyl)piperidine-2-carboxylic acid.

In some embodiments, the immune checkpoint inhibitor is a peptide. See, e.g., Sasikumar et al., "Peptide and Peptide-Inspired Checkpoint Inhibitors: Protein Fragments to Cancer Immunotherapy," Medicine in Drug Discovery 8:100073 (2020).

VI. Mutants In some embodiments, the fusion proteins or components thereof described herein, or the NK cell genotypes described herein, are at least 80%, e.g., at least 85%, 90%, 95%, 98% or 100% identical to the amino acid sequence of the exemplary sequences (e.g., provided herein), e.g., include mutations described herein, or in addition, have up to 1%, 2%, 5%, 10%, 15% or 20% differences as residues of the exemplary sequences replaced, e.g., with conservative mutations. In preferred embodiments, the variants maintain the desired activity of the parent sequence.

To determine the sequence identity of two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps are introduced into one or both of the first amino acid or nucleic acid sequence and the second amino acid or nucleic acid sequence for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. Nucleotides are then compared at corresponding amino acid or nucleotide positions. If a position in the first sequence contains a nucleotide identical to a corresponding position in the second sequence, then the molecules are identical at that position (as used herein, "identity" of nucleic acids is the same as "homology" of nucleic acids). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the length of each gap and the number of gaps that should be introduced to optimally align the two sequences.

The percent identity between a subject polypeptide or nucleic acid sequence (i.e., query) and a second polypeptide or nucleic acid sequence (i.e., target) can be determined in a variety of ways within the skill of the art, for example, using Smith Waterman alignments (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); GeneMatcher Plus ^™ , Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M. O. , Ed, pp 353-358; the BLAST program (Basic Local Alignment Search Tool; (Altschul, S.F., W. Gish, et al. (1990) J Mol. Biol 215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL or Megalign (DNASTAR) software. In conjunction with this, one skilled in the art can determine appropriate parameters for determining alignment, such as any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. In general, for target proteins or nucleic acids, the length of comparison may be any length of the target, up to and including the full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%). For purposes of the present invention, percent identity is based on the full length of the query sequence.

For purposes of the present invention, alignment of two sequences and determination of percent identity can be accomplished using the Blossum62 scoring matrix, applying a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

VII. Definitions Unless otherwise specified, all technical terms, notations, and other technical and scientific terms or terminology used in this application are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined in this application for clarity and/or ease of reference, but the inclusion of such definitions in the present invention should not necessarily be interpreted as meaning a substantial difference from what is commonly understood in the art.

Throughout this application, various embodiments are presented in a range format. This description in range format is primarily for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, whenever possible, a description of a range should be considered to specifically refer to all subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically refer to subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. as well as individual numerical values within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "sample" includes a plurality of samples, including a mixture of samples.

The terms "ascertain," "measure," "assess," "determine," "assay," and "analyze" are used interchangeably herein to refer to types of measurements that are often made. These terms include determining (e.g., detecting) the presence or absence of an element. These terms can encompass quantitative, qualitative, or quantitative and qualitative determinations. Assessment can be relative or absolute. "Detecting the presence of" can encompass determining the amount of something present, other than ascertaining the presence or absence of something, depending on the context.

The terms "individual", "person" or "patient" are often used interchangeably herein.

The term "in vivo" is used to refer to phenomena that take place in an individual's body.

The term "in vitro" is used to indicate a phenomenon that takes place outside an individual's body. An in vitro analysis is not performed on an individual. Rather, it is performed on a sample that is isolated from an individual. An example of an in vitro analysis that is performed on a sample is an "in vitro" analysis.

The term "in vitro" is used to refer to phenomena that take place in a container for holding laboratory reagents such that the material is separated from the biological source from which it is obtained. In vitro analysis can encompass cell-based analysis using living or dead cells. In vitro analysis can also encompass acellular analysis, which does not use intact cells.

As used herein, the term "about" in reference to a number means that 10% of the number in question can be added or subtracted from that number. The term "about" in reference to a range means from the lowest value minus 10% of the range to the highest value plus 10% of the range.

As used herein, the term "buffer solution" refers to an aqueous solution that is a mixture of a weak acid and its counterbase, or vice versa.

As used herein, the term "cell culture medium" refers to a mixture for in vitro cell growth and proliferation that contains factors essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, inorganic substances, etc.

Buffer solutions as used in this application are not cell culture media.

As used herein, the term "bioreactor" refers to a culture device in which a range of conditions that affect cell culture, such as dissolved oxygen concentration, dissolved carbon dioxide concentration, pH and temperature, can be continuously controlled.

As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term encompasses vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are suitable for transferring the nucleic acid molecules or polynucleotides of the invention. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as expression vectors.

The term "operably linked" means that two or more nucleic acid sequences or polypeptide elements are typically physically linked and exist in a functional relationship to each other. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or regulating the transcription or expression of the coding sequence, in which case the coding sequence should be understood to be "under the control of" the promoter.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells encompass "engineered cells," "transformants," and "transformed cells," which encompass the primary engineered (e.g., transformed) cell and its derived progeny, regardless of the number of subcultures. The progeny may not be completely identical in nucleic acid makeup to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as those selected or selected from the originally transformed cell are also included in the invention.

Where appropriate, host cells can be stably or transiently transfected with a polynucleotide encoding a fusion protein as described herein.

The section headings used herein are for organizational purposes only and should not be construed as limiting the scope of the described invention.

VIII. EXAMPLES The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Off-the-shelf NK cell therapy platform An example of a method for expanding and stimulating NK cells is shown in FIG.

High affinity mutants of the receptor CD16 (158V/V mutant, see, for example, Koene et al., "FcγRIIIa-158V/F Polymorphism Influences the Binding of IgG by Natural Killer Cell FcgammaRIIIa, Independently of the FcgammaRIIIa-48L/R/H Phenotype", Blood 90:1109-14 (1997)) and KIR-B genotype (KIR B alleles of the KIR receptor system, see, for example, Hsu et al., "The Killer Cell Immunoglobulin-Like Receptor (KIR) Genomic Region: Gene-Order, Haplotypes and Allelic Polymorphism," Immunological Review 190:40-52 (2002); and Pyo et al., "Different Patterns of Evolution in the Centromeric and Telemeric Regions of Group A and B Haplotypes of the Human Killer Cell Ig-like Receptor Locus," PLoS FDA-approved frozen umbilical cord blood single units with a 5% NK cell number (see One5:e15115 (2010)) were selected as the NK cell source.

Cord blood units were thawed and centrifuged to remove the freezing medium. The cell preparations were then depleted for T cells using the QuadroMACS cell sorting system (Miltenyi) and CD3 (T cell) MicroBeads. A population of ^6x108 total nucleated cells (TNC) were labeled with MicroBeads and separated using the QuadroMACS device and buffer. After T cell depletion, the remaining cells, primarily mononuclear cells and NK cells, were washed and then collected in antibiotic-free medium (CellgroSCGM). The cell preparations were subsequently analyzed for total nucleated cell count, viability, and % CD3+ cells. Cord blood NK cells were CD3 depleted as shown in Figure 1.

CD3- cell preparations were seeded into gas-permeable cell expansion bags with growth medium. Cells were co-cultured with replicatively engineered HuT-78 (eHUT-78) feeder cells to enhance expansion against a master cell bank (MCB) population. CellgroSCGM culture medium was initially supplemented with anti-CD3 antibody (OKT3), human plasma, glutamine, and IL-2.

As shown in FIG. 1, during one of the co-culture steps, the NK cells were selectively engineered to introduce CAR into the NK cells, for example, using a lentiviral vector.

The cells were cultured as static cultures at 37°C in a 5% _CO2 balanced air atmosphere for 12-16 days with additional medium changes at 2-4 day intervals. When the cultures were expanded 100-fold or more, the cultured cells were harvested and then suspended in freezing medium and packed into cryobags. In this case, 80 bags or vials with ¹⁰⁸ cells per bag or vial were produced during such co-culture. The cryobags were frozen in a vapor-phase liquid nitrogen ( _LN2 ) tank at -150°C or below using a controlled rate freezer. Such cryopreserved NK cells derived from FDA-cleared umbilical cord units were used as a master cell bank (MCB).

To produce the drug product, the frozen cell bags obtained from the MCB were thawed and the freezing medium was removed. The thawed cells were inoculated into disposable culture bags and co-cultured with feeder cells, e.g., eHUT78 feeder cells, to produce the drug product. In this case, the cells were cultured in a 50-1 bioreactor to produce several thousand drug products lots per cord blood unit (e.g., 4,000-8,000 cryovials, each with ¹⁰⁹ cells/vial), which were mixed with a cryopreservation composition and then frozen in multiple storage containers, such as cryovials. The drug product is an off-the-shelf infusion ready product available for direct infusion. Each drug product lot can be infused into several hundred to several thousand patients (e.g., 100-1,000 patients, e.g., target dose ^4x109 cells).

Example 2: Amplification of Feeder Cells As an example, suitable feeder cells, e.g., eHut-78 cells, were thawed from frozen stocks and expanded and cultured in 125 ml flasks in culture medium containing RPMI 1640 (Life Technologies) 89% v/v, inactivated fetal bovine serum (FBS) (Life Technologies) (10% v/v) and glutamine (hyclone) (2 mM) at or about 37°C, under 3-7% _CO2 or about 3-7% _CO2 for or about 18-24 days. Cells were split into 125 ml-2 L flasks every 2-3 days. Cells were harvested by centrifugation and gamma irradiated. The harvested and irradiated cells were mixed with cryopreservation medium (Cryostor CS10) in 2 ml cryovials and then frozen in a controlled rate freezer at a cooling rate of about 15°C per 5 min to a final temperature of −90°C or about −90°C, and then transferred to a liquid nitrogen tank or freezer for cooling to a final temperature of −150°C or about −150°C.

After freezing, cell viability was greater than 70% of the original cell number (wherein there were at least 1.0 x ¹⁰⁸ viable cells/ml), greater than 85% of the cells expressed tmTNF-α, greater than 85% of the cells expressed mbIL-21+, and greater than 85% of the cells expressed 4-1BBL.

Example 3: Expansion and stimulation of NK cells As an example, suitable NK cells can be produced using HuT-78 cells transduced to express 4-1BBL, membrane-bound IL-21 and mutant TNFα ("eHut-78P cells") as feeder cells. The feeder cells were suspended in 1% (v/v) CellGro medium and irradiated with 20,000 cGy in a gamma radiation device. Seed cells (e.g., CD3-depleted PBMCs or CD3-depleted umbilical cord blood cells) were cultured on the feeder cells in CellGro medium containing human plasma, glutamine, IL-2 and OKT-3 at 37°C as static cultures. Cells were split at 2-4 day intervals. Total culture time is 19 days. NK cells were harvested by centrifugation and cryopreserved. The thawed NK cells are administered to the patient in an infusion medium composed of: phosphate buffered saline (PBS 1x, FujiFilm Irvine) (50% v/v), albumin (human) (20% v/v OctaPharma albumin solution: human albumin ≧96% protein 200 g/l, sodium 130-160 mmol; ≦2 mmol potassium, 0.064-0.096 mmol/g protein N-acetyl-DL-tryptophan, 0.064-0.096 mmol/g protein, caprylic acid, ad. 1000 ml water), dextran 40/dextrose (25% v/v Hospira Dextran 40/Dextrose Injection, USP containing: 10 g/100 ml dextran 40 and 5 g/100 ml aqueous dextrose solution) and dimethyl sulfoxide (DMSO) (5% v/v Avantor DMSL solution, density 1.101 g/cm ³ , 20° C.).

In some cases, the seed cells are CD3-depleted cord blood cells. The cell fraction can be depleted of CD3 cells by immunomagnetic selection using a CliniMACS T cell depletion set (LS Depletion set (162-01) Miltenyi Biotec).

Preferably, the cord blood cells are selected to express CD16 with V/V polymorphism at F158 (Fc gamma RIIIa-158 V/V genotype) (Musolino et al. 2008 J Clin Oncol 26:1789). Preferably, the cord blood cells are of the KIR-B haplotype.

Example 4: Umbilical cord blood NK cells as a source of NK cells comprise 5-15% of peripheral blood lymphocytes. Traditionally, peripheral blood has been used as a source of NK cells for therapeutic applications. However, as noted herein, umbilical cord blood-derived NK cells have approximately 10-fold higher expansion potential in the culture system described herein compared to peripheral blood-derived methods, and do not experience premature cell exhaustion or senescence. Expression of receptors of interest on the NK cell surface, such as receptors that participate in NK cell activation in tumor cell engagement, has been determined to be more consistent between donors in umbilical cord blood NK cells compared to peripheral blood NK cells. Utilizing the manufacturing process described herein consistently activates NK cells in umbilical cord blood in a donor-independent manner to achieve large-scale production of active and consistent NK cells.

As shown in Figure 2, in preclinical studies, cord blood-derived NK cells (CB-NK) have approximately 10 times higher proliferation in culture than peripheral blood-derived NK cells (PB-NK). As shown in Figure 3, the expression of activating immune receptors on tumor-engaging NK cells was superior and more consistent in cord blood-derived products compared to peripheral blood-engaging.

Example 5: Phenotype of Expanded and Stimulated NK-Cells As an example, NK cells derived from umbilical cord blood units were expanded and stimulated with eHut-78 cells using the expansion and stimulation steps described in Example 1. As shown in Figure 4, the resulting population of expanded and stimulated NK cells consistently exhibited high CD16(158V) and activating NK cell receptor expression.

Example 6: AB-101
AB-101 is a universal, standardized, cryopreserved, allogeneic cord blood-derived NK cell therapy product containing ex vivo expanded and activated effector cells designed to enhance ADCC anti-tumor responses in patients, e.g., patients treated with monoclonal antibodies or NK cell engagers. AB-101 is composed of cord blood-derived mononuclear cells (CBMCs) enriched for NK cells by T lymphocyte depletion and co-cultured with engineered replicating-deficient T cell feeder cells supplemented with IL-2 and anti-CD3 antibody (OKT3).

AB-101 is an FDA-approved cord blood derived allogeneic NK-cell product specifically designed for use in combination with therapeutic monoclonal antibodies (mAbs) to treat hematologic and solid tumors. The AB-101 manufacturing process produces an NK cell product with the following attributes:

- Consistent NK cell profile. High expression of surface receptors such as CD16 and tumor antigen-binding/activating receptors, e.g., NKG2D, NKp46, Nkp30 and NKp44, to which the antibodies bind.

- KIR-B-haplotype. The KIR-B haplotype is associated with improved clinical outcomes in haploidentical transplant settings and higher therapeutic potential in the allogeneic setting.

- CD16 F158V polymorphism. The high affinity CD16 F158V variant that binds mAb Fc appears to promote improved antibody-dependent cellular cytotoxicity (ADCC).

- Non-transformed NK cells. Genetic enhancement or gene editing is not required for or part of the AB-101 drug.

The components and composition of AB-101 are listed in Table 13. AB-101 is composed of NK cells (CD16 ⁺ , CD56 ⁺ ) expressing the natural cytotoxicity receptors NKp30 and NKp46, signifying mature NK cells. AB-101 contains minimal amounts of T cells, B cells and macrophages (≦0.2% CD3 ⁺ , ≦1.0% CD19 ⁺ , ≦1.0% CD14 ⁺ ). The residual percentage of eHuT-78P feeder cells used in the culture of AB-101 is ≦0.2% of the drug product.

Initial stability studies indicate that AB-101 is stable in vapor phase liquid nitrogen for up to six months. Long-term stability studies to evaluate product stability beyond six months are underway and updated stability information will be included in the Certificate of Analysis.

The creation of the AB-101 drug product consists of the following core steps (Figure 5):
Thawing of FDA approved umbilical cord blood units (Hemacord, BLA125937).
Removal of freezing-storage medium from cord blood units (CBUs); CD3 depletion using FDA-cleared Vario MACS cell sorting system (Miltenyi); Expansion and co-culture in bags with engineered feeder cells (eHuT-78 cells); Testing and cryopreservation of AB-101 master cell bank (approximately 200 bags); Thawing (1 bag), amplified and co-culture with engineered HuT-78 cells; Further expansion in bioreactor; Harvesting and filling (1x10 ⁹ NK cells/vial).
・Frozen storage of AB-101 medicine (approximately 150 vials)
Extensive characterization to ensure concentration, purity, potency and stability.

As shown in Table 14, this production process reproducibly produces large quantities of highly pure and active AB-101 therapeutic NK cells. Data points represent results from three independent cord blood units.

Appearance, Suspension AB-101 drug product vials are inspected for appearance by visual observation assessing clarity, color, and the presence or absence of particulates.

Cell counts are determined using the ADAM cell counting system. The ADAM system uses two types of staining solutions: (1) propidium iodide (PI) and cell lysis solution to count total cells; and (2) propidium iodide (PI) and PBS to count non-viable cells. AB-101 drug samples are stained with propidium iodide and loaded onto an Accuchip 4X. The Accuchip is attached to the ADAM cell counting system to determine cell number, cell concentration, and cell viability.

Cell Viability The viability of AB-101 drug products is analyzed using the ADAM cell counting system as described above.

Mycoplasma (USP<63>)
Mycoplasma testing is performed according to the agar and broth media procedures set forth in USP <63>. Aliquots of the AB-101 drug product are added to agar and broth media, respectively. The media are then incubated under aerobic (5% CO ₂ ) conditions for 14 days and anaerobic (5% CO ₂ /N ₂ ) conditions for 28 days as the "broth media test." If the drug substance is contaminated with mycoplasma, colonies should appear on the agar media and a color change should occur in the broth media.

Sterility (USP<71>)
Sterility testing is performed by the "direct inoculation" method described in USP <71>"SterilityTesting". An aliquot of the test sample is transferred directly to a growth-promoting culture medium capable of culturing the microorganism. It is incubated at an appropriate temperature for the recommended period suggested by the USP. After incubation, the growth of the microorganism is visually confirmed.

Endotoxins (USP<85>)
The endotoxin test is performed by the "Kinetic Turbidimetric" method described in USP <85>. Bacterial endotoxins are cell wall components of gram-negative bacteria. The bacterial endotoxin test is an assay used to detect or quantitate endotoxins in gram-negative bacteria. The endotoxin content of the test specimen is determined by reading the results of the test specimen sample dilutions against a standard curve based on the turbidity rate of the lysis reagent that reaches a specific absorbance in the presence of endotoxin.

Karyotype analysis (G-band)
G-banded karyotyping will be performed on the AB-101 drug. The analysis has a maximum resolution of 5-10 megabase pairs. This method should detect balanced and unbalanced translocations.

CNV cytogenetic analysis (high-density SNP array)
Copy Number Variation (CNV) analysis is performed on AB-101 drug by cytogenetic analysis using its high density SNP array to detect copy number variation, duplication/deletion, unbalanced translocation and aneuploidy. To measure CNV, genomic DNA is isolated, quantified, amplified, sheared into fragments and hybridized to a bead chip for analysis. The fluorescence type and intensity of each probe are analyzed by software.

Identity (CD3-, CD56+)
The frequency of CD3-, CD56+ cells is used to analyze the identity of the AB-101 drug product. AB-101 drug product samples are thawed and resuspended in staining buffer. The resuspended samples are added to a fluorophore-labeled antibody that binds to CD3+ and CD56+ surface antigens. Flow cytometry measurements are used to confirm the % CD3-, CD56+ population as a measure of product identity.

Identity (CD56+, CD16+)
The frequency of CD56+, CD16+ cells is used to analyze the identity of the AB-101 drug product. AB-101 drug product samples are thawed and resuspended in staining buffer. The resuspended samples are added to fluorophore-labeled antibodies that bind to CD56+ and CD16+ surface antigens. Flow cytometry measurements are used to confirm the % CD56+, CD16+ population as a measure of product identity.

Purity (CD3+)
The measurement of CD3+ expressing cells is used to assess the purity of the AB-101 drug product. A flow cytometry measurement method is used to determine the purity of CD3+ expressing cells in the drug product. The percentage of the CD3+ cell population is used as a measure of the purity of the product.

Purity (CD14+)
The measurement of CD14+ expressing cells is used to assess the purity of the AB-101 drug product. A flow cytometry measurement method is used to determine the purity of CD14+ expressing cells in the drug product. The % of the population of CD14+ cells is used as a measure of the purity of the product.

Purity (CD19+)
The measurement of CD19+ expressing cells is used to assess the purity of the AB-101 drug product. A flow cytometry measurement method is used to determine the purity of CD19+ expressing cells in the drug product. The percentage of the population of CD19+ cells is used as a measure of the purity of the product.

Purity: eHuT-78P Remaining (eHuT-78P Cell Remaining)
The eHuT-78P cell residues in the AB-101 drug product are measured by flow cytometry (FACS). The eHuT-78 residues in the AB-101 DP are detected by quantifying live CD3+4-1BBLhigh+ eHuT-78P using FACS. As eHuT-78 is derived from the HuT-78 cell line, which expresses CD3 as cutaneous T lymphocytes, a FACS gating strategy (see FIG. 1) is utilized that sequentially gates on singlets, 7-AAD, and CD3+4-1BBL+. The HuT-78 cell line was transduced with 4-1BB ligand (4-1BBL), membrane tumor necrosis factor-α (mTNF-α), and membrane-bound IL-21 (mbIL-21). eHuT-78 single cells that strongly expressed the three genes were selected, and research, master and working cell banks were established sequentially. Among these three genes, 4-1BBL was confirmed to be most strongly expressed in the AB-101 cell bank and the final drug product, and therefore it was used in the FACS gating strategy.

Potency (cytotoxicity in AB-101 DP cells:K562 cells 10:1)
The efficacy of AB-101 drugs is determined by evaluating their cellular cytotoxicity ability against K562 tumor cells. Cytotoxicity of drugs is evaluated by fluorimetric analysis. K562 tumor cells are stained with 30 μM calcein-AM (Molecular probe) for 1 hour at 37°C. Drug samples and labeled tumor cells are co-cultured in triplicate in a 96-well plate at 37°C and 5% _CO2 for 4 hours in the dark. To provide spontaneous maximum release, targets are supplemented with RPMI 1640 medium supplemented with 10% FBS or 2% Triton-X100. To determine background fluorescence, each well is supplemented with RPMI 1640 medium supplemented with 10% FBS or 2% Triton-X100. Fluorescence is measured at excitation 485 nm and emission 535 nm using a fluorescence reader. Specific cytotoxicity % is calculated by the following formula:

Efficacy (cytotoxicity in AB-101 DP cells: Ramos cells 10:1)

The same methods and calculations described above will be used to assess the cellular cytotoxicity potential of the AB-101 drug against Ramos tumor cells to determine efficacy. Details of this test are currently being finalized.

Example 7: Phenotypic characterization of AB-101 The purity as well as the expression of antibody-engaging CD16 and activation, inhibitory and chemokine receptors in various AB-101 batches were determined by flow cytometric measurements.

AB-101 purity was determined using cell surface markers: AB-101 batches were observed to contain >99% CD3-CD56+ NK cells and <0.1% CD3+, CD14+ and CD19+ cells. CD16 expression was measured in AB-101. 95.11±2.51% of AB-101 cells were CD16+, with mean and median MFI for CD16 of 15311±6186 and 13097±5592, respectively. NK cells are known to express a variety of NK-specific activating and inhibitory receptors. In the various AB-101 batches tested, >80% of cells expressed CD16, NKG2A, NKG2D, CD94, NKp30, 2B4, Tim-3, CD44, 40-70% of cells expressed NKp44, NKp46, DNAM-1, approximately 30% of cells expressed CD161 and CD96, 15% of cells expressed CXCR3, and less than 5% of cells expressed other activation inhibitor receptors.

Two GMP batches of AB-101 were included in this study to evaluate the phenotypic characteristics of cells at three different stages of the production process: cord blood cells after CD3+ cell depletion; an intermediate product, the Master Cell Bank (MCB), and AB-101 final drug product (DP). Purity and NK cell receptors were measured for the CD3-depleted cells, MCB, and DP, respectively. The results confirmed that the CB-derived NK cells exhibited an immature NK phenotype. The NK phenotype matured during the production process. At the MCB stage, more than 90% of the cells expressed phenotypic characteristics already present in mature NK cells, with other cell type markers at <0.1%. The expression levels of most of the NK cell-specific receptors increased throughout the production process, from the CD3-depleted cells, to the MCB, and finally to the DP.

Abbreviation list: NK: natural killer cell; mAb: monoclonal antibody; TNF-α: tumor necrosis factor α; CXCR: CXC chemokine receptor; DNAM-1: DNAX accessory molecule-1; CRACC: CD2-like receptor-activated cytotoxicity cells; ILT2: Ig-like transcript 2; Tim-3: T-cell immunoglobulin mucin-3; 7AAD: 7-amino-actinomycin D; ULBP: UL16-binding protein; MICA/B: MHC class I chain-associated proteins A and B; RAE1: ribonucleic acid efflux 1; H60: NKG2D interacts with two cell surface ligands associated with class I MHC molecules; MULT1: mouse UL16-binding protein-like transcript 1; MHC: major histocompatibility complex; HLA: human leukocyte antigen.

Phenotype and purity staining protocol: 1. Adjust the concentration of NK cells to ^2.0x106 cells/ml with cold FACS buffer. 2. Prepare the antibody mixture with reference to the table below. 3. Place the antibody mixture in a 5ml round-bottom tube and mix with 100μl of cell dilution. 4. Stain the cells for 30 minutes at 4°C under light protection. 5. After staining, add 2ml of FACS and centrifuge at 2000rpm and 4°C for 3 minutes. 6. Discard the supernatant and vortex the cell pellet. Then add 200μl of FACS buffer. 7. Analyze the cells with a flow cytometer (LSR Fortessa). 8. Analyze the expression level of each marker using Flow-Jo software. 9. Gate the phenotype with the following gating options: a. Singlet gating with FSC-A/FSC-H panel. b. gating on live cells with 7-AAD/SSC-A panel; c gating on lymphocytes with FSC-A/SSC-A panel; d gating on NK cells (CD3-CD56+) with CD3/CD56; e gating on CD3/CD56, CD16/CD56, and CD14/CD19 after creating quadrants with isotype controls; f gating on PE fluorescence expression (1 and 3-30 in Table 1, % expression) for each marker based on FMO (Fluorescence Minus One) with NK cell gating; for CD16, obtaining ratio mean and median values.

A list of antibody combinations for staining NK cell phenotypes is shown in Table 15.

Purity of AB-101 (n=9)
The purity of AB-101 is expressed as CD3-CD56+ cells for NK cells, CD3+ cells for T cells, CD14+ cells for monocytes, and CD19+ cells for B cells. A total of nine batches of AB-101 were assayed for purity. The results were 99.27±0.59% (mean±SD) CD3-CD56+ cells, 0.02±0.03% CD3+ cells, 0.10±0.12% CD14+ cells, and 0.02±0.04% CD19+ cells (Figure 6). Thus, it was verified that AB-101 is composed of highly pure NK cells with almost no other types of cells present as impurities.

Comparison of Purity of CD3-Depleted Cells, MCB and DP Produced under GMP Conditions Two GMP batches of AB-101 were used to analyze the purity of the AB-101 starting material (CD3-depleted cells), intermediate product (Master Cell Bank, MCB) and final drug product (DP). 50-60% of the cells in the CD3-depleted cell fraction were NK cells, and this percentage increased to over 90% in the MCB and DP. 20-30% of the CD3-depleted cell fraction were CD14+ and CD19+ cells, and this percentage of cells decreased to less than 0.1% in the MCB and DP, indicating the purity of the AB-101 MCB and the final drug product of AB-101 (Figure 7, Table 16).

Comparison of NK cell receptors in CD3-depleted cells, MCB and DP produced under GMP conditions The GMP batch of AB-101 was further used to analyze the expression of various NK cell receptors in the AB-101 starting material (CD3-depleted cells), intermediate product (Master Cell Bank, MCB) and final drug product (DP). It was observed that several NK cell and activating receptors such as CD16, NKG2D, NKG2C, NKp30, NKp44, NKp46 and DNAM-1 were expressed at higher levels in the MCB and final drug product when compared to the AB-101 starting material (CD3-depleted cells). CD57 expression was lower in the MCB and final drug product compared to the AB-101 starting material (CD3-depleted cells) (Figure 8, Table 17). Overall, the data showed increased expression of NK cell activating receptors in the MCB and final drug product, implying that AB-101 is effective against tumors.

Conclusion The use of surface marker analysis demonstrated the identity and purity of the AB-101 product and batch-to-batch consistency. Furthermore, extensive evaluation of NK-specific activating and inhibitory cell surface markers established a consistent profile of the AB-101 product after the production amplification step. CB-derived NK cells have an immature phenotype, such as high expression of NKG2A and low expression of NKG2C, CD62L, CD57, IL-2R, CD16, and DNAM-1, compared to peripheral blood (PB)-derived NK cells, and CB-derived NK cells with an immature phenotype are known to have low cytotoxicity against tumor cells. The data from this experiment demonstrate that the allogeneic cord blood (CB)-derived NK cell product, i.e., AB-101, expresses high levels of key activating receptors, which implies potentially better cytotoxicity against tumor cells.

Example 8: AB-101 Toxicology The non-clinical toxicity of AB-101 was evaluated in a GLP study in NSG mice. The study was designed to evaluate the acute and delayed toxicity profile of AB-101. AB-101 was tested in this study at two dose levels, ^0.5x107 and ^2x107 cells/animal. The proposed test dose range was designed to provide a level of exposure for the product greater than the highest human equivalent dose planned to be provided in the first human study ( ^4x109 cells/dose). Based on allometric scaling (Nair 2016), ^0.5x107 cells/mouse corresponds to ^14x109 cells/human and ^2x107 cells/mouse corresponds to ^56x109 cells/human, which is extrapolated to a patient weighing 70 kg. AB-101 was administered intravenously via the tail vein once weekly for 8 weeks. Acute toxicity of AB-101 was assessed 3 days after the 8th (i.e., final) dose. Delayed toxicity was assessed at the end of a 28-day recovery period after the 8th dose. Survival, body weight, clinical observations, and palpation of each animal were recorded for the life of the study. All animals underwent gross necropsy at the time of euthanasia, with sample collection for blood analysis, clinical chemistry, and histopathology analysis.

Each group consisted of 20 animals, 10 of each sex, to evaluate findings in both sexes and for powered statistical analysis. A vehicle-treated control group was included for comparison with the AB-101-treated groups. To minimize treatment bias, animals were assigned to dose groups by a computer-generated (by weight) random procedure, with females and males randomized separately. The study adhered to GLP guidelines, including data recording.

No mortality or adverse clinical observations were recorded related to administration of AB-101 at any of the dose levels evaluated. All minor observations that emerged were typical of mice and were not considered related to administration of AB-101. Body and organ weight changes were similar between dose groups and at the dates evaluated post-treatment (53 days for the acute toxicity group and 78 days for the delayed toxicity group). There were no AB-101-related changes in hematology and clinical chemistry parameters or gross necropsy findings identified in animals at the time of euthanasia in either the acute or delayed toxicity groups. All variations between individuals and mean clinical chemistry values, regardless of statistical significance, were considered sporadic, consistent with biological and procedure-related deviations and/or minor in size, and therefore unrelated to AB-101 administration. There were no AB-101-related microscopic findings. In conclusion, the results of the GLP toxicity studies suggest that AB-101 is well tolerated in NSG mice following repeated dosing at up to 2×10 ⁷ cells/dose/animal.

Example 9: Cryopreservation of NK cells AB-101 cells were prepared according to the process shown in Figure 5. At the end of the culture period, cells were harvested using a Sartorius ^kSep® 400 disposable automated centrifuge system at a relative centrifugal force (RCF): 800-1200g and a flow rate of 60-120ml/min, and washed twice with phosphate buffered saline (PBS). After washing, ^AB-101 cells were formulated with (1) albumin (human); (2) dextran 40; (3) DMSO; and (4) PBS at a target concentration of 1x108 cells/ml (Cryopreservation Composition Example #1, Table 4 ⁾ . The suspension formulation was filled into 10ml AT-Closed vials® with a target volume of 11ml, then labeled and placed in a controlled rate freezer for cryopreservation at ≦-135°C.

Stability studies were performed at time = 0 for initial release testing data. The stability storage freezer was a validated vapor phase _LN2 storage freezer set to maintain temperature ≦-135°C. At the sterility time point, 10% of the batch size or 4 vials, whichever was greater, was tested. Tests were thawed at 37°C to mimic clinical thawing conditions.

As shown in Table 18, it was confirmed that the viability and activity of cryopreserved AB-101 was preserved for at least 9 months.

A "bedside" short-term stability study was conducted to characterize the stability of AB-101 during handling immediately prior to administration. Samples were thawed, transferred to 10 ml syringes, filtered, and the contents were stored in Falcon tubes at the indicated temperatures for the indicated times. The harvested products were analyzed. Short-term stability data for the two AB-101 lots are shown in Table 19.

Example 10: Cord blood NK cells selected for KIR-B and CD16 158 v/v have low CD38 expression after expansion NK cells were expanded as described in Example 6 to generate AB-101 cells using two cord blood cells from different donors selected for KIR-B and CD16 158 v/v, and one non-selected donor cord blood (control group). The resulting cell purity (CD56+CD3-%) as measured by flow cytometry is shown in Figure 9. As shown in Figures 10 and 11, CD38 expression was lower collectively (% positive, Figure 10) and individually (mean fluorescence intensity of positive cells, Figure 11) in KIR-B/158 v/v NK cells compared to non-selected NK cells.

Example 11: Surface Protein Expression of AB-101 NK cells were expanded as described in Example 6. Surface protein expression of the starting NK cell source (cord blood gated for CD56+/CD3- expression, n=3) was compared to the expanded resulting NK cells (n=16). As shown in Figure 12, CD16 expression was high in the resulting cells and increased compared to the starting cells. Expression of NKG2D, CD94, NKp30, NKp44 and NKp46 was also increased, whereas expression of CXCR4 and CD122 was decreased.

Example 12: ADCC of AB-101 in combination with obinutuzumab
The ADCC of AB-101 cells was examined in combination with obinutuzumab against the cell line ARH-77 tumor cells (ATCC catalog number: CRL-1621) in a 4-hour caspase assay using various concentrations of obinutuzumab and various E:T ratios. ARH-77 cells are a CD20+ plasma cell leukemia cell line. As shown in Figure 13, AB-101 killing activity was increased 66-fold with 1 μg/ml obinutuzumab.

Other embodiments

Although the present invention has been described with detailed description, it should be understood that the above description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims (67)

A method of treating a CD20+ cancer patient comprising administering a population of natural killer cells (NK cells) and an antibody targeting human CD20, the NK cells being allogeneic to the patient, KIR-B haplotype, and homozygous for CD16 158V polymorphism. The method of claim 1, wherein the cancer is non-Hodgkin's lymphoma (NHL). The method of claim 2, wherein the NHL is indolent NHL. The method according to claim 3, wherein the indolent NHL is selected from the group consisting of follicular lymphoma, lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia, gastric MALT, non-gastric MALT, lymph node marginal zone lymphoma, splenic marginal zone lymphoma, small cell lymphocytic lymphoma (SLL) and chronic lymphocytic lymphoma (CLL). The method of claim 4, wherein the small cell lymphocytic lymphoma (SLL) or chronic lymphocytic lymphoma (CLL) includes lymph node or splenic involvement. The method of claim 2, wherein the NHL is an aggressive NHL. The method of claim 6, wherein the aggressive NHL is selected from the group consisting of diffuse large B-cell lymphoma, mantle cell lymphoma, transformed follicular lymphoma, follicular lymphoma (stage IIIB), transformed mucosa-associated lymphoid tissue (MALT) lymphoma, primary mediastinal large B-cell lymphoma, lymphoblastic lymphoma, and high-grade B-cell lymphomas with MYC and BCL2 translocations. The method of claim 7, wherein the aggressive B-cell lymphoma with MYC and BLC2 translocation additionally contains a BCL6 translocation. The method according to any one of claims 1 to 8, wherein the patient relapsed after treatment with an anti-CD20 antibody. The method of any one of claims 1 to 9, wherein the patient has progressed to disease progression following treatment with autologous stem cell transplantation or chimeric antigen receptor T-cell therapy (CAR-T). The method of any one of claims 1 to 10, wherein the patient is administered 1 x 10 ⁸ to 1 x 10 ¹⁰ NK cells. The method of any one of claims 1 to 11, wherein the patient is administered 1 x 10 ⁹ to 8 x 10 ⁹ NK cells. The method of any one of claims 1 to 12, wherein the patient is administered 4x10 ⁸ , 1x10 ⁹ , 4x10 ⁹ or 8x10 ⁹ NK cells. The method according to any one of claims 1 to 13, wherein the antibody is selected from the group consisting of obinutuzumab (or a biosimilar thereof), ofatumumab (or a biosimilar thereof), or a combination thereof. The method of claim 14, wherein the antibody is obinutuzumab. The method of claim 14, wherein the antibody is ofatumumab. The method according to any one of claims 1 to 16, wherein the patient is administered lymphodepleting chemotherapy prior to treatment. The method of claim 17, wherein the lymphodepleting chemotherapy is a non-myeloablative chemotherapy. The method of claim 17 or 18, wherein the lymphodepleting chemotherapy comprises treatment with one or more of cyclophosphamide and fludarabine. 20. The method of claim 19, wherein the lymphodepleting chemotherapy comprises treatment with cyclophosphamide and fludarabine. The method of claim 19 or 20, wherein the cyclophosphamide is administered at 100-500 mg/m ² /day. 22. The method of claim 21, wherein the cyclophosphamide is administered at 250 mg/ ^m2 /day. 22. The method of claim 21, wherein the cyclophosphamide is administered at 500 mg/ ^m2 /day. The method according to any one of claims 19 to 23, wherein the fludarabine is administered at 10-50 mg/m ² /day. 24. The method of claim 23, wherein the fludarabine is administered at 30 mg/ ^m2 /day. The method according to any one of claims 1 to 25, further comprising administration of IL-2. 27. The method of claim 26, wherein the patient is administered IL-2 at 1 x ¹⁰⁶ IU/ ^m2 . The method of claim 26, wherein the patient is administered 6,000,000 IU of IL-2. The method according to any one of claims 26 to 28, wherein the administration of IL-2 is performed within 1-4 hours of administration of the NK cells. The method according to any one of claims 1 to 29, wherein the administration of the NK cells and the human CD20-targeting antibody is performed weekly. The method according to any one of claims 1 to 30, wherein the NK cells and human CD20-targeting antibody are administered weekly for 4-8 weeks. The method according to any one of claims 1 to 31, wherein the NK cells are administered weekly and the human CD20-targeting antibody is administered every other week. The method according to any one of claims 1 to 32, wherein the NK cells are not genetically modified. The method according to any one of claims 1 to 33, wherein the NK cells are at least 70% CD56+ and CD16+. The method of any one of claims 1 to 34, wherein the NK cells are at least 85% CD56+ and CD3-. The method according to any one of claims 1 to 35, wherein 1% or less of the NK cells are CD3+, 1% or less of the NK cells are CD19+, and 1% or less of the NK cells are CD14+. The method of any one of claims 1 to 36, wherein each administration of NK cells is an administration of 1 x 10 ⁹ to 5 x 10 ⁹ NK cells. 35. The method of claim 34, wherein each administration of said NK cells is an administration of 1 x ¹⁰⁹ to 5 x ¹⁰⁹ NK cells. The method of any one of claims 1 to 38, wherein the patient is administered a dose of a CD20-targeted antibody prior to the first administration of NK cells. The method according to any one of claims 1 to 39, wherein the expanded natural killer cells are expanded umbilical cord blood natural killer cells. The method of any one of claims 1 to 40, wherein the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% CD16+ cells. The method of any one of claims 1 to 41, wherein the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKG2D+ cells. The method of any one of claims 1 to 42, wherein the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp46+ cells. The method of any one of claims 1 to 43, wherein the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp30+ cells. The method of any one of claims 1 to 44, wherein the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% DNAM-1+ cells. The method of any one of claims 1 to 45, wherein the expanded population of natural killer cells comprises at least 60%, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% NKp44+ cells. The method of any one of claims 1 to 46, wherein the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD3+ cells. The method of any one of claims 1 to 47, wherein the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD14+ cells. The method of any one of claims 1 to 48, wherein the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD19+ cells. The method of any one of claims 1 to 49, wherein the expanded population of natural killer cells comprises less than 20%, e.g., 10% or less, 5% or less, 1% or less, 0.5% or less, or 0% CD38+ cells. The method according to any one of claims 1 to 50, wherein the natural killer cells do not contain a CD16 transgene. The method according to any one of claims 1 to 51, wherein the natural killer cells do not express exogenous CD16 protein. The method of any one of claims 1 to 52, wherein the expanded natural killer cells are not genetically engineered. The method according to any one of claims 1 to 53, wherein the expanded natural killer cells are derived from the same umbilical cord blood donor. The method of any one of claims 1 to 54, wherein the population of NK cells comprises at least 100 million expanded natural killer cells, e.g., 200 million, 250 million, 300 million, 400 million, 500 million, 600 million, 700 million, 750 million, 800 million, 900 million, 1 billion, 2 billion, 3 billion, 4 billion, 5 billion, 6 billion, 7 billion, 8 billion, 9 billion, 10 billion, 15 billion, 20 billion, 25 billion, 50 billion, 75 billion, 80 billion, 90 billion, 100 billion, 200 billion, 250 billion, 300 billion, 400 billion, 500 billion, 600 billion, 700 billion, 800 billion, 900 billion, 1 trillion, 2 trillion, 3 trillion, 4 trillion, 5 trillion, 6 trillion, 7 trillion, 8 trillion, 9 trillion, or 10 trillion. The method according to any one of claims 1 to 55, wherein the population of NK cells is produced by a method comprising the steps of:
(a) obtaining seed cells comprising natural killer cells from umbilical cord blood;
(b) depleting CD3+ cells with the seed cells;
(c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, a TNFα mutant, and a 4-1BBL gene to produce expanded natural killer cells;
This produces an expanded population of natural killer cells. 57. The method of any one of claims 1 to 56, wherein the population of NK cells is produced by a method comprising:
(a) obtaining seed cells comprising natural killer cells from umbilical cord blood;
(b) depleting CD3+ cells with the seed cells;
(c) expanding the natural killer cells by culturing the depleted seed cells with a first plurality of Hut78 cells engineered to express membrane-bound IL-21, TNFα mutation and the 4-1BBL gene to produce a master cell bank population of expanded natural killer cells; and (d) expanding the master cell bank population of expanded natural killer cells by culturing with a second plurality of Hut78 cells engineered to express membrane-bound IL-21, TNFα mutation and the 4-1BBL gene to produce expanded natural killer cells;
This produces an expanded population of natural killer cells. The population of NK cells, after step (c),
(i) freezing the master cell bank population of amplified natural killer cells in a plurality of containers; and (ii) thawing the containers containing aliquots of the master cell bank population of amplified natural killer cells,
58. The method of claim 56 or 57, wherein in step (d), expanding the master cell bank population of amplified natural killer cells comprises amplifying an aliquot of the master cell bank population of amplified natural killer cells. The method of any one of claims 56 to 58, wherein the cord blood is derived from a donor who is KIR-B haplotype and homozygous for CD16 158V polymorphism. The method according to any one of claims 56 to 59, wherein the population of NK cells is produced by a method comprising amplifying natural killer cells from umbilical cord blood by at least 10,000-fold, e.g., 15,000-fold, 20,000-fold, 25,000-fold, 30,000-fold, 35,000-fold, 40,000-fold, 45,000-fold, 50,000-fold, 55,000-fold, 60,000-fold, 65,000-fold or 70,000-fold. The method of any one of claims 56 to 60, wherein the expanded population of natural killer cells is not enriched or sorted after expansion. The method according to any one of claims 56 to 61, wherein the percentage of NK cells expressing CD16 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood. The method according to any one of claims 56 to 62, wherein the percentage of NKG2D-expressing NK cells in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood. The method according to any one of claims 56 to 63, wherein the percentage of NK cells expressing NKp30 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood. The method according to any one of claims 56 to 64, wherein the percentage of NK cells expressing NKp44 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood. The method according to any one of claims 56 to 65, wherein the percentage of NK cells expressing NKp46 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood. The method of any one of claims 56 to 66, wherein the percentage of NK cells expressing DNAM-1 in the expanded population of natural killer cells is the same as or higher than the percentage of natural killer cells in seed cells derived from umbilical cord blood.